Methods for biological sample processing and analysis

ABSTRACT

Provided are methods for biological sample processing and analysis. A method can comprise providing a substrate configured to rotate. The substrate can comprise an array having immobilized thereto a biological analyte. A solution comprising a plurality of probes may be directed, via centrifugal force, across the substrate during rotation of the substrate, to couple at least one of the plurality of probes with the biological analyte. A detector can be configured to detect a signal from the at least one probe coupled to the biological analyte, thereby analyzing the biological analyte.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/416,889, filed May 20, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/974,364, filed May 8, 2018, which claims thebenefit of U.S. Provisional Patent Application No. 62/588,139, filedNov. 17, 2017, U.S. Provisional Patent Application No. 62/623,743, filedJan. 30, 2018, and U.S. Provisional Patent Application No. 62/664,049,filed Apr. 27, 2018, each of which applications is entirely incorporatedherein by reference.

BACKGROUND

Biological sample processing has various applications in the fields ofmolecular biology and medicine (e.g., diagnosis). For example, nucleicacid sequencing may provide information that may be used to diagnose acertain condition in a subject and in some cases tailor a treatmentplan. Sequencing is widely used for molecular biology applications,including vector designs, gene therapy, vaccine design, industrialstrain design and verification. Biological sample processing may involvea fluidics system and/or a detection system.

SUMMARY

Despite the prevalence of biological sample processing systems andmethods, such systems and methods may have low efficiency that can betime-intensive and wasteful of valuable resources, such as reagents.Recognized herein is a need for methods and systems for sampleprocessing and/or analysis with high efficiency.

The present disclosure provides methods and systems for sampleprocessing and/or analysis.

In an aspect, provided is a method for processing a biological analyte,comprising: (a) providing a substrate comprising an array havingimmobilized thereto the biological analyte, wherein the substrate isrotatable with respect to a central axis; (b) directing a solutioncomprising a plurality of probes across the substrate and in contactwith the biological analyte during rotation of the substrate, whereinthe solution is directed centrifugally along a direction away from thecentral axis; (c) subjecting the biological analyte to conditionssufficient to conduct a reaction between at least one probe of theplurality of probes and the biological analyte, to couple the at leastone probe to the biological analyte; and (d) detecting one or moresignals from the at least one probe coupled to the biological analyte,thereby analyzing the biological analyte.

In some embodiments, the biological analyte is a nucleic acid molecule,and wherein analyzing the biological analyte comprises identifying asequence of the nucleic acid molecule. In some embodiments, theplurality of probes is a plurality of nucleotides. In some embodiments,(c) comprises subjecting the nucleic acid molecule to a primer extensionreaction under conditions sufficient to incorporate at least onenucleotide from the plurality of nucleotides into a growing strand thatis complementary to the nucleic acid molecule. In some embodiments, in(d), the one or more signals are indicative of incorporation of the atleast one nucleotide. In some embodiments, the plurality of nucleotidescomprise nucleotide analogs. In some embodiments, the plurality ofnucleotides is of a first canonical base type. In some embodiments, themethod further comprises repeating (b) and (c) with an additionalplurality of nucleotides that are of a second canonical base type,wherein the second canonical base type is different than the firstcanonical base type. In some embodiments, the plurality of probes is aplurality of oligonucleotide molecules.

In some embodiments, the biological analyte is a nucleic acid molecule,and (c) comprises conducting a complementarity binding reaction betweenthe at least one probe and the nucleic acid molecule to identify apresence of homology between the at least one probe and the biologicalanalyte in (d).

In some embodiments, the detecting in (d) is conducted using a sensorthat continuously scans the array along a nonlinear path during rotationof the substrate.

In some embodiments, the method further comprises, prior to (b), (i)dispensing the solution on the substrate when the substrate isstationary, and (ii) subjecting the substrate to rotation to direct thesolution across the array.

In some embodiments, the method further comprises (i) subjecting thesubstrate to rotation prior to (b), and (ii) while the substrate isrotating, dispensing the solution on the substrate.

In some embodiments, the method further comprises repeating (b)-(d) withan additional plurality of probes that is different than the pluralityof probes.

In some embodiments, the fluid viscosity of the solution or a rotationalvelocity of the substrate is selected to yield a predetermined thicknessof a layer of the solution adjacent to the array.

In some embodiments, the biological analyte is immobilized to the arrayvia a linker.

In some embodiments, the biological analyte is coupled to a bead, whichbead is immobilized to the array.

In some embodiments, the solution is directed to the array using one ormore dispensing nozzles that are directed at or in proximity to thecentral axis of the substrate.

In some embodiments, the array comprises a plurality of individuallyaddressable locations, and wherein the biological analyte is disposed ata given individually addressable location of the plurality ofindividually addressable locations.

In some embodiments, the array has immobilized thereto one or moreadditional biological analytes.

In some embodiments, the substrate is textured or patterned.

In some embodiments, the one or more signals include one or more opticalsignals.

In some embodiments, the method further comprises terminating rotationof the substrate prior to detecting the one or more signals in (d).

In some embodiments, (b) and/or (c) is performed while the substrate isrotated at a first angular velocity and (d) is performed while thesubstrate is rotated at a second angular velocity that is different thanthe first angular velocity.

In some embodiments, the substrate is movable with respect to thecentral axis, and wherein (b) and/or (c) is performed when the substrateis at a first location of the central axis and (d) is performed when thesubstrate is at a second location of the central axis, which secondlocation is different from the first location. In some embodiments, atthe first location the substrate rotates at a first angular velocity andat the second location the substrate rotates at a second angularvelocity that is different than the first angular velocity.

In some embodiments, the array is a substantially planar array.

In another aspect, provided is a method for processing a biologicalanalyte, comprising: (a) providing a substrate comprising asubstantially planar array having immobilized thereto the biologicalanalyte, wherein the substrate is rotatable with respect to a centralaxis; (b) directing a solution comprising a plurality of probes acrossthe substantially planar array and in contact with the biologicalanalyte during rotation of the substrate; (c) subjecting the biologicalanalyte to conditions sufficient to conduct a reaction between at leastone probe of the plurality of probes and the biological analyte, tocouple the at least one probe to the biological analyte; and (d)detecting one or more signals from the at least one probe coupled to thebiological analyte, thereby analyzing the biological analyte.

In some embodiments, the biological analyte is a nucleic acid molecule,and wherein analyzing the biological analyte comprises identifying asequence of the nucleic acid molecule.

In some embodiments, the detecting in (d) is conducted using a sensorthat continuously scans the substantially planar array along a nonlinearpath during rotation of the substrate.

In some embodiments, the substantially planar array comprises aplurality of individually addressable locations, and wherein thebiological analyte is disposed at a given individually addressablelocation of the plurality of individually addressable locations.

In another aspect, provided is a system for analyzing a biologicalanalyte, comprising: a substrate comprising an array configured toimmobilize the biological analyte, wherein the substrate is configuredto rotate with respect to a central axis; a fluid flow unit comprising afluid channel configured to dispense a solution comprising a pluralityof probes to the array, wherein during rotation of the substrate, thesolution is directed centrifugally along a direction away from thecentral axis and brought in contact with the biological analyte underconditions sufficient to couple at least one probe of the plurality ofprobes to the biological analyte; a detector in optical communicationwith the array, wherein the detector is configured to detect one or moresignals from the at least one probe coupled to the biological analyte;and one or more computer processors operatively coupled to the fluidflow unit and the detector, wherein the one or more computer processorsare individually or collectively programmed to (i) direct the fluid flowunit to dispense the solution through the fluid channel to the array,which solution comprising the plurality of probes is directedcentrifugally along a direction away from the central axis and broughtin contact with the biological analyte during rotation of the substrate,and (ii) use the detector to detect the one or more signals from the atleast one probe coupled to the biological analyte.

In some embodiments, the substrate is movable along the central axis. Insome embodiments, the fluid channel is configured to dispense thesolution when the substrate is at a first location along the centralaxis, and wherein the detector is configured to detect the one or moresignals when the substrate is at a second location along the centralaxis, which second location is different from the first location. Insome embodiments, wherein at the first location, the substrate isrotatable at a first angular velocity and, at the second location, thesubstrate is rotatable at a second angular velocity that is differentthan the first angular velocity.

In some embodiments, the system further comprises an additional fluidchannel comprising configured to dispense an additional solution to thearray, wherein the fluid channel and the additional fluid channel arefluidically isolated upstream from one another upstream of outlet portsof the fluid channel and the additional fluid channel.

In some embodiments, the system further comprises an optical imagingobjective configured to be at least partially immersed in a fluid incontact with the substrate, which optical imaging objective is inoptical communication with the detector.

In some embodiments, the system further comprises a container encirclingthe optical imaging objective, which container is configured to retainat least a portion of the fluid.

In some embodiments, the fluid channel does not contact the substrate.

In some embodiments, the array is a substantially planar array.

In some embodiments, the one or more computer processors areindividually or collectively programmed to direct the fluid flow unit todispense the solution through the fluid channel to the array prior torotation of the substrate.

In some embodiments, the one or more computer processors areindividually or collectively programmed to direct the fluid flow unit todispense the solution through the fluid channel to the array when thesubstrate is undergoing rotation.

In some embodiments, the detector is configured to detect the one ormore signals during rotation of the substrate. In some embodiments, thedetector is configured to continuously scan the array along a nonlinearpath during rotation of the substrate.

In some embodiments, the detector is configured to detect the one ormore signals when the substrate is not rotating.

In some embodiments, the detector is an optical detector and wherein theone or more signals are one or more optical signals.

In some embodiments, the array comprises a plurality of individuallyaddressable locations. In some embodiments, the plurality ofindividually addressable locations are individually physicallyaccessible.

In some embodiments, the substrate is textured or patterned.

In some embodiments, the system further comprises a container comprisingthe substrate. In some embodiments, the system further comprises anenvironmental unit that is configured to regulate a temperature or ahumidity of an environment of the container. In some embodiments, thedetector comprises a time delay and integration (TDI) sensor or apseudo-TDI rapid frame rate sensor. In some embodiments, the systemfurther comprises an additional detector in optical communication withthe array, wherein the detector and the additional detector areconfigured to scan the array along different paths. In some embodiments,the different paths are non-linear.

In some embodiments, the system further comprises one or more opticsbetween, and in optical communication with, the array and the detector,wherein the one or more optics are configured to provide an opticalmagnification gradient across the array. In some embodiments, theoptical magnification gradient is anamorphic.

In another aspect, provided is a system for sequencing a nucleic acidmolecule, comprising: a substrate comprising a substantially planararray configured to immobilize a biological analyte, wherein thesubstrate is configured to rotate with respect to a central axis; afluid flow unit comprising a fluid channel configured to dispense asolution comprising a plurality of probes to the substantially planararray, wherein during rotation of the substrate, the solution isdirected across the substantially planar array and brought in contactwith the biological analyte under conditions sufficient to couple atleast one probe of the plurality of probes to the biological analyte; adetector in optical communication with the substantially planar array,wherein the detector is configured to detect one or more signals fromthe at least one probe coupled to the biological analyte; and one ormore computer processors operatively coupled to the fluid flow unit andthe detector, wherein the one or more computer processors areindividually or collectively programmed to (i) direct the fluid flowunit to dispense the solution through the fluid channel to the array,which solution comprising the plurality of probes is directed across thesubstantially planar array and brought in contact with the biologicalanalyte during rotation of the substrate, and (ii) use the detector todetect the one or more signals from the at least one probe coupled tothe biological analyte.

In some embodiments, the system further comprises an optical imagingobjective configured to be at least partially immersed in a fluid incontact with the substrate, which optical imaging objective is inoptical communication with the detector. The fluid may be confined orcontrolled, such as by using an electrical field controlling thehydrophobicity of one or more of regions on the substrate and/or a fluidenclosure.

In some embodiments, the detector comprises a time delay and integration(TDI) sensor or a pseudo-TDI rapid frame rate sensor.

In some embodiments, the detector is configured to detect the one ormore signals during rotation of the substrate. In some embodiments, thedetector is configured to continuously scan the array along a nonlinearpath during rotation of the substrate.

In an aspect, provided is a method for analyte detection or analysis,comprising: (a) rotating an open substrate about a central axis, theopen substrate having an array of immobilized analytes thereon; (b)delivering a solution having a plurality of probes to a region proximalto the central axis to introduce the solution to the open substrate; (c)dispersing the solution across the open substrate at least bycentrifugal force such that at least one of the plurality of probesbinds to at least one of the immobilized analytes to form a bound probe;and (d) using a detector to detect at least one signal from the boundprobe via continuous rotational area scanning of the open substrate.

In some embodiments, the continuous rotational area scanning compensatesfor velocity differences at different radial positions of the array withrespect to the central axis within a scanned area. In some embodiments,the continuous rotational area scanning comprises using an opticalimaging system having an anamorphic magnification gradient substantiallytransverse to a scanning direction along the open substrate, and whereinthe anamorphic magnification gradient at least partially compensates fortangential velocity differences that are substantially perpendicular tothe scanning direction. In some embodiments, the continuous rotationalarea scanning comprises reading two or more regions on the opensubstrate at two or more scan rates, respectively, to at least partiallycompensate for tangential velocity differences in the two or moreregions.

In some embodiments, (d) further comprises using an immersion objectivelens in optical communication with the detector and the open substrateto detect the at least one signal, which immersion objective lens is incontact with a fluid that is in contact with the open substrate. In someembodiments, the fluid is in a container, and an electric field is usedto regulate a hydrophobicity of one or more surfaces of the container toretain at least a portion of the fluid contacting the immersionobjective lens and the open substrate.

In some embodiments, the continuous rotational area scanning isperformed in a first environment having a first operating condition, andwherein the delivering of the solution is performed in a secondenvironment having a second operating condition different from the firstoperating condition.

In some embodiments, the immobilized analytes comprise nucleic acidmolecules, wherein the plurality of probes comprises fluorescentlylabeled nucleotides, and wherein at least one of the fluorescentlylabeled nucleotides binds to at least one of the nucleic acid moleculesvia nucleotide complementarity binding.

In some embodiments, the open substrate is substantially planar.

In another aspect, provided is an apparatus for analyte detection oranalysis, comprising: a housing configured to receive an open substratehaving an array of immobilized analytes thereon; one or more dispensersconfigured to deliver a solution having a plurality of probes to aregion proximal to a central axis of the open substrate; a rotationalunit configured to rotate the open substrate about a central axis tothereby disperse the solution across the open substrate at least bycentrifugal force, such that at least one of the plurality of probesbinds to at least one of the analytes to form a bound probe; and adetector configured to detect at least one signal from the bound probevia continuous rotational area scanning of the open substrate.

In some embodiments, the detector is configured to compensate forvelocity differences at different radial positions of the array withrespect to the central axis within a scanned area. In some embodiments,the one or more optics are configured to generate an anamorphicmagnification gradient substantially transverse to a scanning directionalong the open substrate, and wherein the anamorphic magnificationgradient at least partially compensates for tangential velocitydifferences that are substantially perpendicular to the scanningdirection. In some embodiments, the apparatus further comprises aprocessor configured to adjust the anamorphic magnification gradient tocompensate for different imaged radial positions with respect to thecentral axis.

In some embodiments, the detector is configured to scan two or moreregions on the open substrate at two or more scan rates, respectively,to at least partially compensate for tangential velocity differences inthe two or more regions.

In some embodiments, the detector comprises a sensor and one or moreoptics in optical communication with the open substrate.

In some embodiments, the apparatus further comprises an immersionobjective lens in optical communication with the detector and the opensubstrate, which immersion objective lens is configured to be in contactwith a fluid that is in contact with the open substrate. In someembodiments, the apparatus further comprises a container configured toretain the fluid and an electric field application unit configured toregulate a hydrophobicity of one or more surfaces of the container toretain at least a portion of the fluid contacting the immersionobjective lens and the open substrate. In some embodiments, theimmersion objective lens is configured to separate a first environmentfrom a second environment, wherein the first environment and secondenvironment have different operating conditions. In some embodiments,the immersion objective lens forms a seal between the first environmentand the second environment.

In some embodiments, the detector is configured to detect the at leastone signal from the bound probe in a non-linear scanning path across theopen substrate. In some embodiments, non-linear scanning path is asubstantially spiral scanning path or a substantially ring-like scanningpath.

In another aspect, provided is a computer-readable medium comprisingnon-transitory instructions stored thereon, which when executed causeone or more computer processors to implement a method for analytedetection or analysis, the method comprising: rotating an open substrateabout a central axis, the open substrate having an array of immobilizedanalytes thereon; delivering a solution having a plurality of probes toa region proximal to the central axis, to introduce the solution to theopen substrate; dispersing the solution across the open substrate atleast by centrifugal force such that at least one of the plurality ofprobes binds to at least one of the immobilized analytes to form a boundprobe; and using a detector to detect at least one signal from the boundprobe via continuous rotational area scanning of the open substrate.

In some embodiments, the method further comprises using an immersionobjective lens in optical communication with the detector and the opensubstrate to detect the at least one signal, which immersion objectivelens is in contact with a fluid that is in contact with the opensubstrate. In some embodiments, the method further comprises using anelectric field to regulate a hydrophobicity of one or more surfaces of acontainer to retain at least a portion of the fluid contacting theimmersion objective lens and the open substrate.

In some embodiments, the immobilized analytes comprise nucleic acidmolecules, wherein the plurality of probes comprises fluorescentlylabeled nucleotides, and wherein at least one of the fluorescentlylabeled nucleotides binds to at least one of the nucleic acid moleculesvia a primer extension reaction.

In some embodiments, the continuous rotational area scanning compensatesfor velocity differences at different radial positions of the array withrespect to the central axis within a scanned area. In some embodiments,the continuous rotational area scanning comprises using an opticalimaging system having an anamorphic magnification gradient substantiallytransverse to a scanning direction along the open substrate, and whereinthe anamorphic magnification gradient at least partially compensates fortangential velocity differences that are substantially perpendicular tothe scanning direction. In some embodiments, the method furthercomprises adjusting the anamorphic magnification gradient to compensatefor different imaged radial positions with respect to the central axis.In some embodiments, the detector is configured to scan two or moreregions on the open substrate at two or more scan rates, respectively,to at least partially compensate for tangential velocity differences inthe two or more imaged regions.

In some embodiments, the continuous rotational area scanning comprisesusing an algorithmic compensation for velocity differences substantiallyperpendicular to a scanning direction along the open substrate.

In some embodiments, the detector is configured to detect the at leastone signal from the bound probe in a non-linear scanning path across theopen substrate.

In another aspect, provided is a method for sequencing a nucleic acidmolecule, comprising: (a) providing a substrate comprising a planararray having immobilized thereto the nucleic acid molecule, wherein thesubstrate is configured to rotate with respect to an axis; (b) directinga solution comprising a plurality of nucleotides across the planar arrayduring rotation of the substrate; (c) subjecting the nucleic acidmolecule to a primer extension reaction under conditions sufficient toincorporate at least one nucleotide from the plurality of nucleotidesinto a growing strand that is complementary to the nucleic acidmolecule; and (d) detecting a signal indicative of incorporation of theat least one nucleotide, thereby sequencing the nucleic acid molecule.

The method may further comprise, prior to (b), (i) dispensing thesolution on the substrate when the substrate is stationary, and (ii)subjecting the substrate to rotation to direct the solution across theplanar array. The method may further comprise (i) subjecting thesubstrate to rotation prior to (b), and (ii) while the substrate isrotating, dispensing the solution on the substrate. The method mayfurther comprise repeating (b)-(d) one or more times to identify one ormore additional signals indicative of incorporation of one or moreadditional nucleotides, thereby sequencing the nucleic acid molecule.

Different solutions may be directed to the planar array during rotationof the substrate for consecutive cycles. The rotation may yieldcentrifugal forces that subject the solution to flow over the planararray. A layer thickness of the planar array may be engineered based onadjusting fluid viscosity. A first fluid having a first viscosity may beused for generating a layer with the nucleic acid molecule on the planararray and a second fluid having a second viscosity may be used forwashing the planar array. The first viscosity may be different from thesecond viscosity. The first viscosity may be controlled by controlling atemperature of the first fluid. The second viscosity may be controlledby controlling a temperature of the second fluid.

The planar array may comprise a linker that is coupled to the nucleicacid sample. The nucleic acid sample may be coupled to a bead, whichbead is immobilized to the planar array.

The planar array may be in fluid communication with at least one sampleinlet and at least one sample outlet. The solution may be directed tothe planar array using one or more dispensing nozzles. The one or morenozzles may be directed at or in proximity to a center of the substrate.

The method may further comprise recycling a subset of the solution thathas contacted the substrate. Recycling may comprise collecting,filtering, and reusing the subset of the solution. The filtering may bemolecular filtering.

The planar array may comprise a plurality of individually addressablelocations. The planar array may be textured. The planar array may be apatterned array.

The signal may be an optical signal. The signal may be a fluorescentsignal.

The method may further comprise terminating rotation of the substrateprior to detecting the signal in (d). The signal in (d) may be detectedwhile the substrate is rotating.

The operations (b) and/or (c) may be performed at a first a location and(d) may be performed at a second location that is different from thefirst location. The first location may comprise a first processing bayand the second location may comprise a second processing bay that isdifferent from the second location. The first location may comprise afirst rotating spindle interior to a second rotating spindle and thesecond location may comprise the second rotating spindle. The firstlocation may comprise a first rotating spindle exterior to a secondrotating spindle and the second location may comprise the secondrotating spindle. The first rotating spindle and second rotating spindlemay be configured to rotate at different angular velocities. Theoperation (b) may be performed at the first location. The operation (c)may be performed at the second location. The operation (c) may beperformed at the first location.

The method may further comprise transferring the substrate between thefirst location and the second location. The operations (b) and/or (c)may be performed while the substrate is rotated at a first angularvelocity and (d) may be performed while the substrate is rotated at asecond angular velocity that is different from the first angularvelocity. The first angular velocity may be less than the second angularvelocity. The first angular velocity may be between 0 revolutions perminute (rpm) and 100 rpm. The second angular velocity may be between 100rpm and 5,000 rpm. The operation (b) may be performed while thesubstrate is rotated at the first angular velocity. The operation (c)may be performed while the substrate is rotated at the second angularvelocity. The operation (c) may be performed while the substrate isrotated at the first angular velocity.

In an aspect, a method for sequencing a nucleic acid molecule maycomprise: (a) providing a substrate comprising an array havingimmobilized thereto the nucleic acid molecule, wherein the substrate isconfigured to rotate with respect to an axis; (b) directing a solutioncomprising a plurality of natural nucleotides and/or non-naturalnucleotides across the array during rotation of the substrate; (c)subjecting the nucleic acid molecule to a primer extension reactionunder conditions sufficient to incorporate at least one nucleotide fromthe plurality of natural nucleotides and non-natural nucleotides into agrowing strand that is complementary to the nucleic acid molecule; and(d) detecting a signal indicative of incorporation of the at least onenucleotide, thereby sequencing the nucleic acid molecule.

The method may further comprise, prior to (b), (i) dispensing thesolution on the substrate when the substrate is stationary, and (ii)subjecting the substrate to rotation to direct the solution to thearray. The method may further comprise (i) subjecting the substrate torotation prior to (b), and (ii) while the substrate is rotating,dispensing the solution on the substrate. The method may furthercomprise, subsequent to (c), modifying the at least one nucleotide. Themodifying may comprise labeling the at least one nucleotide. The atleast one nucleotide may be cleavably labeled. The method may furthercomprise, subsequent to (d), cleaving or modifying a label of the atleast one nucleotide. The method may further comprise repeating (b)-(d)one or more times to identify one or more additional signals indicativeof incorporation of one or more additional nucleotides, therebysequencing the nucleic acid molecule.

Different solution may be directed to the array during rotation of thesubstrate for consecutive cycles. Subsequent to (d), and prior to a nextiteration of (b), the at least one nucleotide may be modified. Therotation may yield centrifugal forces that subject the solution to flowover the array. A layer thickness of the array may be engineered basedon fluid viscosity. A first fluid having a first viscosity may be usedfor generating a layer with the nucleic acid molecule on the array and asecond fluid having a second viscosity may be used for washing thearray. The first viscosity may be different from the second viscosity.The first viscosity may be controlled by controlling a temperature ofthe first fluid. The second viscosity may be controlled by controlling atemperature of the second fluid.

The array may comprise a linker that is coupled to the nucleic acidsample. The nucleic acid sample may be coupled to a bead, which bead isimmobilized to the array.

The array may be in fluid communication with at least one sample inletand at least one sample outlet. The solution may be directed to thearray using one or more dispensing nozzles. The one or more nozzles maybe directed at or in proximity to a center of the substrate.

The method may further comprise recycling a subset of the solution thathas contacted the substrate. Recycling may comprise collecting,filtering, and reusing the subset of the solution. The filtering may bemolecular filtering.

The array may comprise a plurality of individually addressablelocations. The array may be planar. The array may be textured. The arraymay be a patterned array.

The signal may be an optical signal. The signal may be a fluorescentsignal.

The method may further comprise, prior to (b), subjecting the substrateto rotation with respect to the axis. The method may further compriseterminating rotation of the substrate prior to detecting the signal in(d). The signal in (d) may be detected while the substrate is rotating.

The operations (b) and/or (c) may be performed at a first a location and(d) may be performed at a second location that is different from thefirst location. The first location may comprise a first processing bayand the second location may comprise a second processing bay that isdifferent from the first processing bay. The first location may comprisea first rotating spindle interior to a second rotating spindle and thesecond location may comprise the second rotating spindle. The firstlocation may comprise a first rotating spindle exterior to a secondrotating spindle and the second location may comprise the secondrotating spindle. The first rotating spindle and second rotating spindlemay be configured to rotate at different angular velocities. Theoperation (b) may be performed at the first location. The operation (c)may be performed at the second location. The operation (c) may beperformed at the first location.

The method may further comprise transferring the substrate between thefirst location and the second location. The operations (b) and/or (c)may be performed while the substrate is rotated at a first angularvelocity and (d) may be performed while the substrate is rotated at asecond angular velocity that is different from the first angularvelocity. The first angular velocity may be less than the second angularvelocity. The first angular velocity may be between 0 rpm and 100 rpm.The second angular velocity may be between 100 rpm and 5,000 rpm. Theoperation (b) may be performed while the substrate is rotated at thefirst angular velocity. The operation (c) may be performed while thesubstrate is rotated at the second angular velocity. The operation (c)may be performed while the substrate is rotated at the first angularvelocity.

In an aspect, a system for sequencing a nucleic acid molecule maycomprise: a substrate comprising an array configured to immobilize thenucleic acid molecule, wherein the substrate is configured to (i) rotatewith respect to an axis and (ii) undergo a change in relative positionwith respect to a longitudinal axis; a first fluid channel comprising afirst fluid outlet port that is configured to dispense a first fluid tothe array; a second fluid channel comprising a second fluid outlet portthat is configured to dispense a second fluid to the array, wherein thefirst fluid channel and the second fluid channel are fluidicallyisolated upstream of the first fluid outlet port; and a detectorconfigured to detect a signal from the array.

The first fluid outlet port and the second fluid outlet port may beexternal to the substrate. The first fluid outlet port and the secondfluid outlet port may not contact the substrate. The first fluid outletport and the second fluid outlet port may be nozzles.

The axis may be substantially parallel with the longitudinal axis. Thelongitudinal axis may be coincident with the axis. The longitudinal axismay be substantially perpendicular to a surface of the substrate. Therelative position of the substrate may be configured to alternatebetween at least a first position and a second position with respect tothe longitudinal axis.

The system may further comprise (i) a third fluid channel comprising afirst fluid inlet port located at a first level of the longitudinalaxis, wherein the first fluid inlet port is downstream of and in fluidcommunication with the substrate when the substrate is in the firstrelative position, and (ii) a fourth fluid channel comprising a secondfluid inlet port located at a second level of the longitudinal axis,wherein the second fluid inlet port is downstream of and in fluidcommunication with the substrate when the substrate is in the relativesecond position. The third fluid channel may be in fluid communicationwith the first fluid channel and the fourth fluid channel may be influid communication with the second fluid channel. The substrate may beconfigured to have (i) the first relative position prior to, during, orsubsequent to receiving the first fluid from the first fluid outlet portand (ii) the second relative position prior to, during, or subsequent toreceiving the second fluid from the second fluid outlet port. The thirdfluid channel and the first fluid channel may define at least part of afirst cyclic fluid flow path and the fourth fluid channel and the secondfluid channel may define at least part of a second cyclic fluid flowpath. At least one of the first cyclic fluid flow path and the secondcyclic fluid flow path may comprise a filter. The filter may be amolecular filter.

The system may further comprise a shield that prevents fluidcommunication between the substrate and (i) the second fluid inlet portwhen the substrate is in the first position and (ii) the first fluidinlet port when the substrate is in the second position. The substratemay be translatable along the longitudinal axis. The substrate may bestationary along the longitudinal axis. At least one of a first axis ofthe first fluid outlet port and a second axis of the second fluid outletport may be substantially coincident with the axis. At least one of afirst axis of the first fluid outlet port and a second axis of thesecond fluid outlet port may be substantially parallel to the axis.

The first fluid and the second fluid may comprise different types ofreagents. The first fluid may comprise a first type of nucleotide ornucleotide mixture and the second fluid may comprise a second type ofnucleotide or nucleotide mixture . The first fluid or the second fluidmay comprise a washing reagent.

The detector may be configured to detect the signal from the substrateduring rotation of the substrate. The detector may be configured todetect the signal from the substrate when the substrate is not rotating.

The signal may be an optical signal. The signal may be a fluorescentsignal.

The first fluid outlet port may be configured to dispense the firstfluid to the array during rotation of the substrate. The second fluidoutlet port may be configured to dispense the second fluid to the arrayduring rotation of the substrate. The first fluid outlet port and thesecond fluid outlet port may be configured to dispense atnon-overlapping times. The substrate may be configured to rotate with atleast one of (i) different speeds and (ii) different number of rotationswhen the first fluid outlet port dispenses and when the second fluidoutlet port dispenses. During the rotation, the array may be configuredto direct the first fluid in a substantially radial direction away fromthe axis. The first fluid outlet port may be configured to dispense thefirst fluid to the array during more than one full rotation of thesubstrate.

The array may comprise a plurality of individually addressablelocations. The array may comprise a plurality of individuallyaddressable locations. The array may comprise a linker that is coupledto the nucleic acid sample. The nucleic acid sample may be coupled to abead, which bead is immobilized to the array. The array may be textured.The array may be a patterned array. The array may be planar.

In an aspect, a system for sequencing a nucleic acid molecule maycomprise: a substrate comprising a planar array configured to immobilizethe nucleic acid molecule, wherein the substrate is configured to rotatewith respect to an axis; a fluid flow unit configured to direct asolution comprising a plurality of nucleotides to the planar arrayduring rotation of the substrate; a detector in sensing communicationwith the planar array; and one or more computer processors operativelycoupled to the fluid flow unit and the detector, wherein the one or morecomputer processors are individually or collectively programmed to (i)direct the fluid flow unit to direct the solution comprising theplurality of nucleotides across the planar array during rotation of thesubstrate; (ii) subject the nucleic acid molecule to a primer extensionreaction under conditions sufficient to incorporate one or morenucleotides from the plurality of nucleotides into a growing strand thatis complementary to the nucleic acid molecule; and (iii) use thedetector to detect one or more signals indicative of incorporation ofthe at one or more nucleotides, thereby sequencing the nucleic acidmolecule.

In an aspect, a system for sequencing a nucleic acid molecule maycomprise: a substrate comprising an array configured to immobilize thenucleic acid molecule, wherein the substrate is configured to rotatewith respect to an axis; a fluid flow unit configured to direct asolution comprising a plurality of nucleotides to the array duringrotation of the substrate, wherein the plurality of nucleotidescomprises natural nucleotides and/or non-natural nucleotides; a detectorin sensing communication with the planar array; and one or more computerprocessors operatively coupled to the fluid flow unit and the detector,wherein the one or more computer processors are individually orcollectively programmed to (i) direct the fluid flow unit to direct thesolution comprising the plurality of nucleotides across the array duringrotation of the array; (ii) subject the nucleic acid molecule to aprimer extension reaction under conditions sufficient to incorporate oneor more nucleotides of the plurality of nucleotides into a growingstrand that is complementary to the nucleic acid molecule; and (iii) usethe detector to detect one or more signals indicative of incorporationof the one or more nucleotides, thereby sequencing the nucleic acidmolecule.

In an aspect, an optical system for continuous area scanning of asubstrate during rotational motion of the substrate, wherein therotational motion is with respect to an axis of the substrate, maycomprise: a focal plane segmented into a plurality of regions; one ormore sensors in optical communication with the plurality of regions; anda controller operatively coupled to the one or more sensors, wherein thecontroller is programmed to process optical signals from each region ofthe plurality of regions with independent clocking during the rotationalmotion, wherein the independent clocking is based at least in part on adistance of each region from a projection of the axis and an angularvelocity of the rotational motion.

The focal plane may be segmented into the plurality of regions along anaxis substantially normal to a projected direction of the rotationalmotion. The focal plane may be segmented into the plurality of regionsalong an axis parallel to a projected direction of the rotationalmotion. The focal plane may be optically segmented.

A given sensor of the one or more sensors may be configured to processeach region of the plurality of regions with independent clocking duringthe rotational motion. The one or more sensors may be a plurality ofsensors, wherein each of the plurality of sensors is in opticalcommunication with a different region of the plurality of regions, andwherein the controller is configured to process optical signals fromeach of the plurality of regions with independent clocking during therotational motion. The one or more sensors may comprise one or more timedelay and integration (TDI), pseudo-TDI rapid frame rate, charge coupleddevice (CCD), or complementary metal oxide semiconductor (CMOS)detectors. The independent clocking may comprise TDI line rate orpseudo-TDI frame rate.

One or more of the sensors may be configured to be in opticalcommunication with at least 2 of the plurality of regions in the focalplane. One or more of the sensors may comprise a plurality of segments.Each segment of the plurality of segments may be in opticalcommunication with a region of the plurality of regions. Each segment ofthe plurality of segments may be independently clocked. The independentclocking of a segment may correspond to a velocity of an image in anassociated region of the focal plane.

The optical system may further comprise an optical imaging objectiveconfigured to be immersed in a fluid. The optical system may furthercomprise an enclosure encircling the optical imaging objective. Theoptical system may further comprise a fluidic line coupled to theenclosure, the fluidic line configured to provide a fluid to theenclosure. The fluid may be in contact with the substrate. The fluid maybe confined or controlled, such as by using an electrical fieldcontrolling the hydrophobicity of one or more of regions on thesubstrate and/or a fluid enclosure.

In an aspect, an optical system for imaging a substrate duringrotational motion of the substrate, wherein the rotational motion iswith respect to an axis of the support, may comprise: a sensor; and anoptical element in optical communication with the sensor, wherein theoptical element is configured to direct optical signals from thesubstrate to the sensor, and wherein at least one of the sensor and theoptical element is configured to generate an optical magnificationgradient across the detector along a direction substantiallyperpendicular to a projected direction of the rotational motion. Thesystem may further comprise a controller operatively coupled to thedetector and the optical element, wherein the controller is programmedto direct adjustment of at least one of the sensor and the opticalelement to generate the optical magnification gradient across the sensoralong the direction substantially perpendicular to a projected directionof the rotational motion.

The optical element may be a lens. The controller may be programmed todirect adjustment of at least one of the sensor and the optical elementto produce an anamorphic optical magnification gradient. A ratio of (i)a first optical magnification at a first radial position of a fielddimension having a least distance in the field dimension from aprojection of the axis to (ii) a second optical magnification at asecond radial position of the field dimension having a greatest distancein the field dimension from the projection of the axis may besubstantially equal to a ratio of the greatest distance to the leastdistance. The optical magnification gradient may be generated byrotation of the optical element and a focal plane substantiallyperpendicular to the projected direction of the rotational motion. Thecontroller may be programmed to direct rotation of the optical element.The controller may be programmed to direct adjustment the gradient ofmagnification based at least in part on a radial range of a fielddimension relative to a projection of the axis. The controller may beprogrammed to subject the rotational motion to the substrate.

The optical system may further comprise an optical imaging objectiveconfigured to be immersed in a fluid. The optical system may furthercomprise an enclosure encircling the optical imaging objective. Theoptical system may further comprise a fluidic line coupled to theenclosure, the fluidic line configured to provide a fluid to theenclosure. The fluid may be in contact with the substrate.

In an aspect, an optical system for imaging a substrate duringrotational motion of the substrate, wherein the rotational motion iswith respect to an axis of the support, may comprise: a plurality ofsensors, each sensor of the plurality of sensors in opticalcommunication with the substrate; and a controller operatively coupledto each sensor of the plurality of sensors, wherein the controller isprogrammed to direct each sensor of the plurality of sensors along animaging path, wherein an imaging path for one or more sensors of theplurality of sensors is distinct from an imaging path of another sensorof the plurality of sensors. The controller may be programmed to directeach sensor of the plurality of sensors along an imaging path having aspiral shape or a ring shape. Each sensor of the plurality of sensorsmay be configured to receive light having a wavelength in apredetermined wavelength range.

The optical system may further comprise an optical imaging objectiveconfigured to be immersed in a fluid. The optical system may furthercomprise an enclosure encircling the optical imaging objective. Theoptical system may further comprise a fluidic line coupled to theenclosure, the fluidic line configured to provide a fluid to theenclosure.

In an aspect, a method for processing an analyte may comprise: (a)providing a substrate comprising a planar array having immobilizedthereto said analyte, wherein said substrate is configured to rotatewith respect to an axis; (b) directing a solution comprising a pluralityof adaptors across said planar array during rotation of said substrate;(c) subjecting said analyte to conditions sufficient to cause a reactionbetween said analyte and said plurality of adaptors; and (d) detecting asignal indicative of said reaction between said analyte and saidplurality of adaptors, thereby analyzing said analyte.

The planar array may comprise two or more types of analytes. The two ormore types of analytes may be arranged randomly. The two or more typesof analytes may be arranged in a regular pattern. The analyte may be asingle cell analyte. The analyte may be a nucleic acid molecule . Theanalyte may be a protein molecule. The analyte may be a single cell. Theanalyte may be a particle. The analyte may be an organism. The analytemay be part of a colony. The analyte may be immobilized in anindividually addressable location on the planar array.

The plurality of adaptors may comprise a plurality of probes. A givenprobe of the plurality of probes may be oligonucleotides 1 to 10 basesin length. A given probe may be a dibase probe. A given probe may be 10to 20 bases in length. The plurality of probes may be labeled.

The substrate may comprise a linker that is coupled to the analyte. Thelinker may comprise a carbohydrate molecule. The linker may comprise anaffinity binding protein. The linker may be hydrophilic. The linker maybe hydrophobic. The linker may be electrostatic. The linker may belabeled. The linker may be integral to the substrate. The linker may bean independent layer on the substrate.

The method may further comprise, prior to (a), directing the analyteacross the substrate comprising the linker. The analytic may be coupledto a bead, which bead is immobilized to the planar array. The planararray may be in fluid communication with at least one sample inlet andat least one sample outlet. The solution may be directed to the planararray using one or more dispensing nozzles. The one or more nozzles maybe directed at or in proximity of the center of the substrate.

The method may further comprise recycling a subset of the solution thathas contacted the substrate. The recycling may comprise collecting,filtering, and reusing the subset of the solution. The filtering may bemolecular filtering.

The planar array may comprise a plurality of individually addressablelocations. The planar array may be textured. The planar array may be apatterned array.

The signal may be an optical signal. The signal may be a fluorescencesignal. The signal may be a light absorption signal. The signal may be alight scattering signal. The signal may be a luminescent signal. Thesignal may be a phosphorescence signal. The signal may be an electricalsignal. The signal may be an acoustic signal. The signal may be amagnetic signal.

The method may further comprise, prior to (b), subjecting the substrateto rotation with respect to the axis. The method may further compriseterminating rotation of the substrate prior to detecting the signal in(d). The signal may be detected in (d) while the substrate is rotating.

The signal may be generated by binding of a label to the analyte. Thelabel may be bound to a molecule, particle, cell, or organism. The labelmay be bound to the molecule, particle, cell, or organism prior to (a).The label may be bound to the molecule, particle, cell, or organismsubsequent to (a). The signal may be generated by formation of adetectable product by a chemical reaction. The reaction may comprise anenzymatic reaction. The signal may be generated by formation of adetectable product by physical association. The signal may be generatedby formation of a detectable product by proximity association. Theproximity association may comprise Förster resonance energy transfer(FRET). The proximity association may comprise association with acomplementation enzyme. The signal may be generated by a singlereaction. The signal may be generated by a plurality of reactions. Theplurality of reactions may occur in series. The plurality of reactionsmay occur in parallel. The plurality of reactions may comprise one ormore repetitions of a reaction. The reaction may comprise ahybridization reaction or ligation reaction. The reaction may comprise ahybridization reaction and a ligation reaction.

The plurality of adaptors may comprise a plurality of carbohydratemolecules. The plurality of adaptors may comprise a plurality of lipidmolecules. The plurality of adaptors may comprise a plurality ofaffinity binding proteins. The plurality of adaptors may comprise aplurality of aptamers. The plurality of adaptors may comprise aplurality of antibodies. The plurality of adaptors may be hydrophilic.The plurality of adaptors may be hydrophobic. The plurality of adaptorsmay be electrostatic. The plurality of adaptors may be labeled. Theplurality of adaptors may comprise a plurality of oligonucleotidemolecules. The plurality of adaptors may comprise a random sequence. Theplurality of adaptors may comprise a targeted sequence. The plurality ofadaptors may comprise a repeating sequence. The repeating sequence maybe a homopolymer sequence.

The method may further comprise repeating (b)-(d) one or more times.Different solutions may be directed to the planar array during rotationof the substrate for consecutive cycles.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows a computer control system that is programmed or otherwiseconfigured to implement methods provided herein;

FIG. 2 shows a method for sequencing a nucleic acid molecule;

FIG. 3 shows a system for sequencing a nucleic acid molecule;

FIG. 4A shows a system for sequencing a nucleic acid molecule in a firstvertical level;

FIG. 4B shows a system for sequencing a nucleic acid molecule in asecond vertical level;

FIG. 5A shows a first example of a system for sequencing a nucleic acidmolecule using an array of fluid flow channels;

FIG. 5B shows a second example of a system for sequencing a nucleic acidmolecule using an array of fluid flow channels;

FIG. 6 shows a computerized system for sequencing a nucleic acidmolecule;

FIG. 7 shows an optical system for continuous area scanning of asubstrate during rotational motion of the substrate;

FIG. 8A shows an optical system for imaging a substrate duringrotational motion of the substrate using tailored optical distortions;

FIG. 8B shows an example of induced tailored optical distortions using acylindrical lens;

FIG. 9A shows a first example of an interleaved spiral imaging scan;

FIG. 9B shows a second example of an interleaved imaging scan;

FIG. 9C shows an example of a nested imaging scan;

FIG. 10 shows a configuration for a nested circular imaging scan;

FIG. 11 shows a cross-sectional view of an immersion optical system;

FIG. 12A shows an architecture for a system comprising a stationary axissubstrate and moving fluidics and optics;

FIG. 12B shows an architecture for a system comprising a translatingaxis substrate and stationary fluidics and optics;

FIG. 12C shows an architecture for a system comprising a plurality ofstationary substrates and moving fluidics and optics;

FIG. 12D shows an architecture for a system comprising a plurality ofmoving substrates on a rotary stage and stationary fluidics and optics;

FIG. 12E shows an architecture for a system comprising a plurality ofstationary substrates and moving optics;

FIG. 12F shows an architecture for a system comprising a plurality ofmoving substrates and stationary fluidics and optics;

FIG. 12G shows an architecture for a system comprising a plurality ofsubstrates moved between a plurality of processing bays;

FIG. 12H shows an architecture for a system comprising a plurality ofimaging heads scanning with shared translation and rotational axes andindependently rotating fields;

FIG. 12I shows an architecture for a system comprising multiple spindlesscanning with a shared optical detection system;

FIG. 13 shows an architecture for a system comprising a plurality ofrotating spindles;

FIG. 14 shows a flowchart for a method for processing an analyte;

FIG. 15 shows a first example of a system for isolating an analyte; and

FIG. 16 shows a second example of a system for isolating an analyte.

FIG. 17 shows examples of control systems to compensate for velocitygradients during scanning.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “processing an analyte,” as used herein, generally refers toone or more stages of interaction with one more sample substances.Processing an analyte may comprise conducting a chemical reaction,biochemical reaction, enzymatic reaction, hybridization reaction,polymerization reaction, physical reaction, any other reaction, or acombination thereof with, in the presence of, or on, the analyte.Processing an analyte may comprise physical and/or chemical manipulationof the analyte. For example, processing an analyte may comprisedetection of a chemical change or physical change, addition of orsubtraction of material, atoms, or molecules, molecular confirmation,detection of the presence of a fluorescent label, detection of a Forsterresonance energy transfer (FRET) interaction, or inference of absence offluorescence. The term “analyte” may refer to molecules, cells,biological particles, or organisms. In some instances, a molecule may bea nucleic acid molecule, antibody, antigen, peptide, protein, or otherbiological molecule obtained from or derived from a biological sample.An analyte may originate from, and/or be derived from, a biologicalsample, such as from a cell or organism. An analyte may be synthetic.

The term “sequencing,” as used herein, generally refers to a process forgenerating or identifying a sequence of a biological molecule, such as anucleic molecule. Such sequence may be a nucleic acid sequence, whichmay include a sequence of nucleic acid bases. Sequencing may be singlemolecule sequencing or sequencing by synthesis, for example. Sequencingmay be performed using template nucleic acid molecules immobilized on asupport, such as a flow cell or one or more beads.

The term “biological sample,” as used herein, generally refers to anysample from a subject or specimen. The biological sample can be a fluidor tissue from the subject or specimen. The fluid can be blood (e.g.,whole blood), saliva, urine, or sweat. The tissue can be from an organ(e.g., liver, lung, or thyroid), or a mass of cellular material, suchas, for example, a tumor. The biological sample can be a feces sample,collection of cells (e.g., cheek swab), or hair sample. The biologicalsample can be a cell-free or cellular sample. Examples of biologicalsamples include nucleic acid molecules, amino acids, polypeptides,proteins, carbohydrates, fats, or viruses. In an example, a biologicalsample is a nucleic acid sample including one or more nucleic acidmolecules, such as deoxyribonucleic acid (DNA) and/or ribonucleic acid(RNA). The nucleic acid molecules may be cell-free or cell-free nucleicacid molecules, such as cell free DNA or cell free RNA. The nucleic acidmolecules may be derived from a variety of sources including human,mammal, non-human mammal, ape, monkey, chimpanzee, reptilian, amphibian,avian, or plant sources. Further, samples may be extracted from varietyof animal fluids containing cell free sequences, including but notlimited to blood, serum, plasma, vitreous, sputum, urine, tears,perspiration, saliva, semen, mucosal excretions, mucus, spinal fluid,amniotic fluid, lymph fluid and the like. Cell free polynucleotides maybe fetal in origin (via fluid taken from a pregnant subject), or may bederived from tissue of the subject itself.

The term “subject,” as used herein, generally refers to an individualfrom whom a biological sample is obtained. The subject may be a mammalor non-mammal. The subject may be an animal, such as a monkey, dog, cat,bird, or rodent. The subject may be a human. The subject may be apatient. The subject may be displaying a symptom of a disease. Thesubject may be asymptomatic. The subject may be undergoing treatment.The subject may not be undergoing treatment. The subject can have or besuspected of having a disease, such as cancer (e.g., breast cancer,colorectal cancer, brain cancer, leukemia, lung cancer, skin cancer,liver cancer, pancreatic cancer, lymphoma, esophageal cancer or cervicalcancer) or an infectious disease. The subject can have or be suspectedof having a genetic disorder such as achondroplasia, alpha-1 antitrypsindeficiency, antiphospholipid syndrome, autism, autosomal dominantpolycystic kidney disease, Charcot-Marie-tooth, cri du chat, Crohn'sdisease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome,Duchenne muscular dystrophy, factor V Leiden thrombophilia, familialhypercholesterolemia, familial Mediterranean fever, fragile x syndrome,Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly,Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonicdystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta,Parkinson's disease, phenylketonuria, Poland anomaly, porphyria,progeria, retinitis pigmentosa, severe combined immunodeficiency, sicklecell disease, spinal muscular atrophy, Tay-Sachs, thalassemia,trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGRsyndrome, or Wilson disease.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidsequence,” “nucleic acid fragment,” “oligonucleotide” and“polynucleotide,” as used herein, generally refer to a polynucleotidethat may have various lengths, such as either deoxyribonucleotides ordeoxyribonucleic acids (DNA) or ribonucleotides or ribonucleic acids(RNA), or analogs thereof. Non-limiting examples of nucleic acidsinclude DNA, RNA, genomic DNA or synthetic DNA/RNA or coding ornon-coding regions of a gene or gene fragment, loci (locus) defined fromlinkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branchednucleic acids, plasmids, vectors, isolated DNA of any sequence, andisolated RNA of any sequence. A nucleic acid molecule can have a lengthof at least about 10 nucleic acid bases (“bases”), 20 bases, 30 bases,40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40kb, 50 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 1 megabase (Mb), ormore. A nucleic acid molecule (e.g., polynucleotide) can comprise asequence of four natural nucleotide bases: adenine (A); cytosine (C);guanine (G); and thymine (T) (uracil (U) for thymine (T) when thepolynucleotide is RNA). A nucleic acid molecule may include one or morenonstandard nucleotide(s), nucleotide analog(s) and/or modifiednucleotide(s).

Nonstandard nucleotides, nucleotide analogs, and/or modified analogs mayinclude, but are not limited to, diaminopurine, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-i sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl adenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine, ethynyl nucleotide bases, 1-propynyl nucleotidebases, azido nucleotide bases, phosphoroselenoate nucleic acids and thelike. In some cases, nucleotides may include modifications in theirphosphate moieties, including modifications to a triphosphate moiety.Additional, non-limiting examples of modifications include phosphatechains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8,9, 10 or more phosphate moieties), modifications with thiol moieties(e.g., alpha-thio triphosphate and beta-thiotriphosphates) ormodifications with selenium moieties (e.g., phosphoroselenoate nucleicacids). Nucleic acid molecules may also be modified at the base moiety(e.g., at one or more atoms that typically are available to form ahydrogen bond with a complementary nucleotide and/or at one or moreatoms that are not typically capable of forming a hydrogen bond with acomplementary nucleotide), sugar moiety or phosphate backbone. Nucleicacid molecules may also contain amine -modified groups, such asaminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) toallow covalent attachment of amine reactive moieties, such asN-hydroxysuccinimide esters (NHS). Alternatives to standard DNA basepairs or RNA base pairs in the oligonucleotides of the presentdisclosure can provide higher density in bits per cubic mm, highersafety (resistant to accidental or purposeful synthesis of naturaltoxins), easier discrimination in photo-programmed polymerases, or lowersecondary structure. Nucleotide analogs may be capable of reacting orbonding with detectable moieties for nucleotide detection.

The term “nucleotide,” as used herein, generally refers to anynucleotide or nucleotide analog. The nucleotide may be naturallyoccurring or non-naturally occurring. The nucleotide analog may be amodified, synthesized or engineered nucleotide. The nucleotide analogmay not be naturally occurring or may include a non-canonical base. Thenaturally occurring nucleotide may include a canonical base. Thenucleotide analog may include a modified polyphosphate chain (e.g.,triphosphate coupled to a fluorophore). The nucleotide analog maycomprise a label. The nucleotide analog may be terminated (e.g.,reversibly terminated). The nucleotide analog may comprise analternative base.

The terms “amplifying,” “amplification,” and “nucleic acidamplification” are used interchangeably and generally refer togenerating one or more copies of a nucleic acid or a template. Forexample, “amplification” of DNA generally refers to generating one ormore copies of a DNA molecule. Moreover, amplification of a nucleic acidmay be linear, exponential, or a combination thereof. Amplification maybe emulsion based or may be non-emulsion based. Non-limiting examples ofnucleic acid amplification methods include reverse transcription, primerextension, polymerase chain reaction (PCR), ligase chain reaction (LCR),helicase-dependent amplification, asymmetric amplification, rollingcircle amplification, recombinase polymerase reaction (RPA), andmultiple displacement amplification (MDA). Where PCR is used, any formof PCR may be used, with non-limiting examples that include real-timePCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR,emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hotstart PCR, inverse PCR, methylation-specific PCR, miniprimer PCR,multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetricinterlaced PCR and touchdown PCR. Moreover, amplification can beconducted in a reaction mixture comprising various components (e.g., aprimer(s), template, nucleotides, a polymerase, buffer components,co-factors, etc.) that participate or facilitate amplification. In somecases, the reaction mixture comprises a buffer that permits contextindependent incorporation of nucleotides. Non-limiting examples includemagnesium-ion, manganese-ion and isocitrate buffers. Additional examplesof such buffers are described in Tabor, S. et al. C.C. PNAS, 1989, 86,4076-4080 and U.S. Pat. Nos. 5,409,811 and 5,674,716, each of which isherein incorporated by reference in its entirety.

Useful methods for clonal amplification from single molecules includerolling circle amplification (RCA) (Lizardi et al., Nat. Genet.19:225-232 (1998), which is incorporated herein by reference), bridgePCR (Adams and Kron, Method for Performing Amplification of Nucleic Acidwith Two Primers Bound to a Single Solid Support, Mosaic Technologies,Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research,Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000);Pemov et al., Nucl. Acids Res. 33:e11(2005); or U.S. Pat. No. 5,641,658,each of which is incorporated herein by reference), polony generation(Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra etal., Anal. Biochem. 320:55-65(2003), each of which is incorporatedherein by reference), and clonal amplification on beads using emulsions(Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), whichis incorporated herein by reference) or ligation to bead-based adapterlibraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenneret al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000)); Reinartz, etal., Brief Funct. Genomic Proteomic 1:95-104 (2002), each of which isincorporated herein by reference).

The term “detector,” as used herein, generally refers to a device thatis capable of detecting a signal, including a signal indicative of thepresence or absence of one or more incorporated nucleotides orfluorescent labels. The detector may detect multiple signals. The signalor multiple signals may be detected in real-time during, substantiallyduring a biological reaction, such as a sequencing reaction (e.g.,sequencing during a primer extension reaction), or subsequent to abiological reaction. In some cases, a detector can include opticaland/or electronic components that can detect signals. The term“detector” may be used in detection methods. Non-limiting examples ofdetection methods include optical detection, spectroscopic detection,electrostatic detection, electrochemical detection, acoustic detection,magnetic detection, and the like. Optical detection methods include, butare not limited to, light absorption, ultraviolet-visible (UV-vis) lightabsorption, infrared light absorption, light scattering, Rayleighscattering, Raman scattering, surface-enhanced Raman scattering, Miescattering, fluorescence, luminescence, and phosphorescence.Spectroscopic detection methods include, but are not limited to, massspectrometry, nuclear magnetic resonance (NMR) spectroscopy, andinfrared spectroscopy. Electrostatic detection methods include, but arenot limited to, gel based techniques, such as, for example, gelelectrophoresis. Electrochemical detection methods include, but are notlimited to, electrochemical detection of amplified product afterhigh-performance liquid chromatography separation of the amplifiedproducts.

The term “continuous area scanning,” as used herein, generally refers toarea scanning in rings, spirals, or arcs on a rotating substrate usingan optical imaging system and a detector. Continuous area scanning mayscan a substrate or array along a nonlinear path. Alternatively or inaddition, continuous area scanning may scan a substrate or array along alinear or substantially linear path. The detector may be a continuousarea scanning detector. The scanning direction may be substantially θ inan (R, θ) coordinate system in which the object rotation motion is in aθ direction. Across any field of view on the object (substrate) imagedby a scanning system, the apparent velocity may vary with the radialposition (R) of the field point on the object as R_(dt/dθ). Continuousarea scanning detectors may scan at the same rate for all imagepositions and therefore may not be able to operate at the correct scanrate for all imaged points in a curved (or arcuate or non-linear) scan.Therefore the scan may be corrupted by velocity blur for imaged fieldpoints moving at a velocity different than the scan velocity. Continuousrotational area scanning may comprise an optical detection system ormethod that makes algorithmic, optical, and/or electronic corrections tosubstantially compensate for this tangential velocity blur, therebyreducing this scanning aberration. For example, the compensation isaccomplished algorithmically by using an image processing algorithm thatdeconvolves differential velocity blur at various image positionscorresponding to different radii on the rotating substrate to compensatefor differential velocity blur.

In another example, the compensation is accomplished by using ananamorphic magnification gradient. This may serve to magnify thesubstrate in one axis (anamorphic magnification) by different amounts attwo or more substrate positions transverse to the scan direction. Theanamorphic magnification gradient may modify the imaged velocities ofthe two or more positions to be substantially equal thereby compensatingfor tangential velocity differences of the two positions on thesubstrate. This compensation may be adjustable to account for differentvelocity gradients across the field of view at different radii on thesubstrate.

The imaging field of view may be segmented into two or more regions,each of which can be electronically controlled to scan at a differentrate. These rates may be adjusted to the mean projected object velocitywithin each region. The regions may be optically defined using one ormore beam splitters or one or more mirrors. The two or more regions maybe directed to two or more detectors. The regions may be defined assegments of a single detector.

The term “continuous area scanning detector,” as used herein, generallyrefers to an imaging array sensor capable of continuous integration overa scanning area wherein the scanning is electronically synchronized tothe image of an object in relative motion. A continuous area scanningdetector may comprise a time delay and integration (TDI) charge coupleddevice (CCD), Hybrid TDI, or complementary metal oxide semiconductor(CMOS) pseudo TDI.

The term “open substrate”, as used herein, generally refers to asubstantially planar substrate in which a single active surface isphysically accessible at any point from a direction normal to thesubstrate. Substantially planar may refer to planarity at a micrometerlevel or nanometer level. Alternatively, substantially planar may referto planarity at less than a nanometer level or greater than a micrometerlevel (e.g., millimeter level).

The term “anamorphic magnification”, as used herein, generally refers todifferential magnification between two axes of an image. An anamorphicmagnification gradient may comprise differential anamorphicmagnification in a first axis across a displacement in the second axis.The magnification in the second axis may be unity or any other valuethat is substantially constant over the field.

The term “field of view”, as used herein, generally refers to the areaon the sample or substrate that is optically mapped to the active areaof the detector.

Processing an Analyte Using a Rotating Array

Prior microfluidic systems have utilized substrates containing numerouslong, narrow channels. The typical flow cell geometry for suchsubstrates introduces a need to compromise between two competingrequirements: 1) minimizing volume to minimize reagent usage; and 2)maximizing effective hydraulic diameter to minimize flow time. Thistrade-off may be especially important for washing operations, which mayrequire large wash volumes and thus long amounts of time to complete.The tradeoff is illustrated by the Poiseuille equation that dictatesflow in the laminar regime and is thus inherent to microfluidic systemsthat utilize such flow cell geometries. Such flow cell geometries mayalso be susceptible to contamination. Because such flow cell geometriesallow for a finite, limited number of channels in the microfluidicsystems, such finite number of channels may be shared between aplurality of different mixtures comprising different analytes, reagents,agents, and/or buffers. Contents of fluids flowing through the samechannels may be contaminated.

Described herein are devices, systems, and methods for processinganalytes using open substrates or flow cell geometries that can addressat least the abovementioned problems. The devices, systems and methodsmay be used to facilitate any application or process involving areaction or interaction between an analyte and a fluid (e.g., a fluidcomprising reagents, agents, buffers, other analytes, etc.). Suchreaction or interaction may be chemical (e.g., polymerase reaction) orphysical (e.g., displacement). The systems and methods described hereinmay benefit from higher efficiency, such as from faster reagent deliveryand lower volumes of reagents required per surface area. The systems andmethods described herein may avoid contamination problems common tomicrofluidic channel flow cells that are fed from multiport valves whichcan be a source of carryover from one reagent to the next. The devices,systems, and methods may benefit from shorter completion time, use offewer resources (e.g., various reagents), and/or reduced system costs.The open substrates or flow cell geometries may be used to process anyanalyte, such as but not limited to, nucleic acid molecules, proteinmolecules, antibodies, antigens, cells, and/or organisms, as describedherein. The open substrates or flow cell geometries may be used for anyapplication or process, such as, but not limited to, sequencing bysynthesis, sequencing by ligation, amplification, proteomics, singlecell processing, barcoding, and sample preparation, as described herein.

The systems and methods may utilize a substrate comprising an array(such as a planar array) of individually addressable locations. Eachlocation, or a subset of such locations, may have immobilized thereto ananalyte (e.g., a nucleic acid molecule, a protein molecule, acarbohydrate molecule, etc.). For example, an analyte may be immobilizedto an individually addressable location via a support, such as a bead. Aplurality of analytes immobilized to the substrate may be copies of atemplate analyte. For example, the plurality of analytes may havesequence homology. In other instances, the plurality of analytesimmobilized to the substrate may be different. The plurality of analytesmay be of the same type of analyte (e.g., a nucleic acid molecule) ormay be a combination of different types of analytes (e.g., nucleic acidmolecules, protein molecules, etc.). The substrate may be rotatableabout an axis. The analytes may be immobilized to the substrate duringrotation. Reagents (e.g., nucleotides, antibodies, washing reagents,enzymes, etc.) may be dispensed onto the substrate prior to or duringrotation (for instance, spun at a high rotational velocity) of thesubstrate to coat the array with the reagents and allow the analytes tointeract with the reagents. For example, when the analytes are nucleicacid molecules and when the reagents comprise nucleotides, the nucleicacid molecules may incorporate or otherwise react with (e.g.,transiently bind) one or more nucleotides. In another example, when theanalytes are protein molecules and when the reagents compriseantibodies, the protein molecules may bind to or otherwise react withone or more antibodies. In another example, when the reagents comprisewashing reagents, the substrate (and/or analytes on the substrate) maybe washed of any unreacted (and/or unbound) reagents, agents, buffers,and/or other particles.

High speed coating across the substrate may be achieved via tangentialinertia directing unconstrained spinning reagents in a partially radialdirection (that is, away from the axis of rotation) during rotation, aphenomenon commonly referred to as centrifugal force. High speedrotation may involve a rotational speed of at least 1 revolution perminute (rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least20 rpm, at least 50 rpm, at least 100 rpm, at least 200 rpm, at least500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, atleast 10,000 rpm, or greater. This mode of directing reagents across asubstrate may be herein referred to as centrifugal or inertial pumping.One or more signals (such as optical signals) may be detected from adetection area on the substrate prior to, during, or subsequent to, thedispensing of reagents to generate an output. For example, the outputmay be an intermediate or final result obtained from processing of theanalyte. Signals may be detected in multiple instances. The dispensing,rotating, and/or detecting operations, in any order (independently orsimultaneously), may be repeated any number of times to process ananalyte. In some instances, the substrate may be washed (e.g., viadispensing washing reagents) between consecutive dispensing of thereagents.

Provided herein is a method for processing a biological analyte,comprising providing a substrate comprising an array having immobilizedthereto the biological analyte, wherein the substrate is rotatable withrespect to a central axis. In some instances, the array can be a planararray. In some instances, the array can be an array of wells. In someinstances, the substrate can be textured and/or patterned. The methodcan comprise directing a solution across the substrate and bringing thesolution in contact with the biological analyte during rotation of thesubstrate. The solution may be directed in a radial direction (e.g.,outwards) with respect to the substrate to coat the substrate andcontact the biological analytes immobilized to the array. In someinstances, the solution may comprise a plurality of probes. In someinstances, the solution may be a washing solution. The method cancomprise subjecting the biological analyte to conditions sufficient toconduct a reaction between at least one probe of the plurality of probesand the biological analyte. The reaction may generate one or moresignals from the at least one probe coupled to the biological analyte.The method can comprise detecting one or more signals, thereby analyzingthe biological analyte.

The substrate may be a solid substrate. The substrate may entirely orpartially comprise one or more of glass, silicon, a metal such asaluminum, copper, titanium, chromium, or steel, a ceramic such astitanium oxide or silicon nitride, a plastic such as polyethylene (PE),low-density polyethylene (LDPE), high-density polyethylene (HDPE),polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrilebutadiene styrene (ABS), polyacetylene, polyamides, polycarbonates,polyesters, polyurethanes, polyepoxide, polymethyl methacrylate (PMMA),polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamineformaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK),polyetherimide (PEI), polyimides, polylactic acid (PLA), furans,silicones, polysulfones, any mixture of any of the preceding materials,or any other appropriate material. The substrate may be entirely orpartially coated with one or more layers of a metal such as aluminum,copper, silver, or gold, an oxide such as a silicon oxide (Si_(x)O_(y),where x, y may take on any possible values), a photoresist such as SU8,a surface coating such as an aminosilane or hydrogel, polyacrylic acid,polyacrylamide dextran, polyethylene glycol (PEG), or any combination ofany of the preceding materials, or any other appropriate coating. Theone or more layers may have a thickness of at least 1 nanometer (nm), atleast 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500μm, or at least 1 millimeter (mm). The one or more layers may have athickness that is within a range defined by any two of the precedingvalues.

The substrate may have the general form of a cylinder, a cylindricalshell or disk, a rectangular prism, or any other geometric form. Thesubstrate may have a thickness (e.g., a minimum dimension) of at least100 μm, at least 200 μm, at least 500 μm, at least 1 mm, at least 2 mm,at least 5 mm, or at least 10 mm. The substrate may have a thicknessthat is within a range defined by any two of the preceding values. Thesubstrate may have a first lateral dimension (such as a width for asubstrate having the general form of a rectangular prism or a radius fora substrate having the general form of a cylinder) of at least 1 mm, atleast 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000mm. The substrate may have a first lateral dimension that is within arange defined by any two of the preceding values. The substrate may havea second lateral dimension (such as a length for a substrate having thegeneral form of a rectangular prism) or at least 1 mm, at least 2 mm, atleast 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. Thesubstrate may have a second lateral dimension that is within a rangedefined by any two of the preceding values. A surface of the substratemay be planar. Alternatively or in addition to, a surface of thesubstrate may be textured or patterned. For example, the substrate maycomprise grooves, troughs, hills, and/or pillars. The substrate maydefine one or more cavities (e.g., micro-scale cavities or nano-scalecavities). The substrate may have a regular textures and/or patternsacross the surface of the substrate. For example, the substrate may haveregular geometric structures (e.g., wedges, cuboids, cylinders,spheroids, hemispheres, etc.) above or below a reference level of thesurface. Alternatively, the substrate may have irregular textures and/orpatterns across the surface of the substrate. For example, the substratemay have any arbitrary structure above or below a reference level of thesubstrate. In some instances, a texture of the substrate may comprisestructures having a maximum dimension of at most about 100%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of thesubstrate or a layer of the substrate. In some instances, the texturesand/or patterns of the substrate may define at least part of anindividually addressable location on the substrate. A textured and/orpatterned substrate may be substantially planar.

The substrate may comprise an array. For instance, the array may belocated on a lateral surface of the substrate. The array may be a planararray. The array may have the general shape of a circle, annulus,rectangle, or any other shape. The array may comprise linear and/ornon-linear rows. The array may be evenly spaced or distributed. Thearray may be arbitrarily spaced or distributed. The array may haveregular spacing. The array may have irregular spacing. The array may bea textured array. The array may be a patterned array. The array maycomprise a plurality of individually addressable locations. The analyteto be processed may be immobilized to the array. The array may compriseone or more binders described herein, such as one or more physical orchemical linkers or adaptors, that are coupled to a biological analyte.For instance, the array may comprise a linker or adaptor that is coupledto a nucleic acid molecule. Alternatively or in addition to, thebiological analyte may be coupled to a bead; the bead may be immobilizedto the array.

The individually addressable locations may comprise locations ofanalytes or groups of analytes that are accessible for manipulation. Themanipulation may comprise placement, extraction, reagent dispensing,seeding, heating, cooling, or agitation. The extraction may compriseextracting individual analytes or groups of analytes. For instance, theextraction may comprise extracting at least 2, at least 5, at least 10,at least 20, at least 50, at least 100, at least 200, at least 500, orat least 1,000 analytes or groups of analytes. Alternatively or inaddition to, the extraction may comprise extracting at most 1,000, atmost 500, at most 200, at most 100, at most 50, at most 20, at most 10,at most 5, or at most 2 analytes or groups of analytes. The manipulationmay be accomplished through, for example, localized microfluidic, pipet,optical, laser, acoustic, magnetic, and/or electromagnetic interactionswith the analyte or its surroundings.

The array may be coated with binders. For instance, the array may berandomly coated with binders. Alternatively, the array may be coatedwith binders arranged in a regular pattern (e.g., in linear arrays,radial arrays, hexagonal arrays etc.). The array may be coated withbinders on at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% of the number ofindividually addressable locations, or of the surface area of thesubstrate. The array may be coated with binders on a fraction ofindividually addressable locations, or of the surface areas of thesubstrate, that is within a range defined by any two of the precedingvalues. The binders may be integral to the array. The binders may beadded to the array. For instance, the binders may be added to the arrayas one or more coating layers on the array.

The binders may immobilize biological analytes through non-specificinteractions, such as one or more of hydrophilic interactions,hydrophobic interactions, electrostatic interactions, physicalinteractions (for instance, adhesion to pillars or settling withinwells), and the like. The binders may immobilize biological analytesthrough specific interactions. For instance, where the biologicalanalyte is a nucleic acid molecule, the binders may compriseoligonucleotide adaptors configured to bind to the nucleic acidmolecule. Alternatively or in addition, such as to bind other types ofanalytes, the binders may comprise one or more of antibodies,oligonucleotides, aptamers, affinity binding proteins, lipids,carbohydrates, and the like. The binders may immobilize biologicalanalytes through any possible combination of interactions. For instance,the binders may immobilize nucleic acid molecules through a combinationof physical and chemical interactions, through a combination of proteinand nucleic acid interactions, etc. The array may comprise at leastabout 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000or more binders. Alternatively or in addition, the array may comprise atmost about 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000,100, 10 or fewer binders. The array may have a number of binders that iswithin a range defined by any two of the preceding values. In someinstances, a single binder may bind a single biological analyte (e.g.,nucleic acid molecule). In some instances, a single binder may bind aplurality of biological analytes (e.g., plurality of nucleic acidmolecules). In some instances, a plurality of binders may bind a singlebiological analyte. Though examples herein describe interactions ofbinders with nucleic acid molecules, the binders may immobilize othermolecules (such as proteins), other particles, cells, viruses, otherorganisms, or the like.

In some instances, each location, or a subset of such locations, mayhave immobilized thereto an analyte (e.g., a nucleic acid molecule, aprotein molecule, a carbohydrate molecule, etc.). In other instances, afraction of the plurality of individually addressable location may haveimmobilized thereto an analyte. A plurality of analytes immobilized tothe substrate may be copies of a template analyte. For example, theplurality of analytes (e.g., nucleic acid molecules) may have sequencehomology. In other instances, the plurality of analytes immobilized tothe substrate may not be copies. The plurality of analytes may be of thesame type of analyte (e.g., a nucleic acid molecule) or may be acombination of different types of analytes (e.g., nucleic acidmolecules, protein molecules, etc.).

In some instances, the array may comprise a plurality of types ofbinders, such as to bind different types of analytes. For example, thearray may comprise a first type of binders (e.g., oligonucleotides)configured to bind a first type of analyte (e.g., nucleic acidmolecules), and a second type of binders (e.g., antibodies) configuredto bind a second type of analyte (e.g., proteins), and the like. Inanother example, the array may comprise a first type of binders (e.g.,first type of oligonucleotide molecules) to bind a first type of nucleicacid molecules and a second type of binders (e.g., second type ofoligonucleotide molecules) to bind a second type of nucleic acidmolecules, and the like. For example, the substrate may be configured tobind different types of analytes in certain fractions or specificlocations on the substrate by having the different types of binders inthe certain fractions or specific locations on the substrate.

A biological analyte may be immobilized to the array at a givenindividually addressable location of the plurality of individuallyaddressable locations. An array may have any number of individuallyaddressable locations. For instance, the array may have at least 1, atleast 2, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, at least 10,000, at least 20,000, at least 50,000, at least100,000, at least 200,000, at least 500,000, at least 1,000,000, atleast 2,000,000, at least 5,000,000, at least 10,000,000, at least20,000,000, at least 50,000,000, at least 100,000,000, at least200,000,000, at least 500,000,000, at least 1,000,000,000, at least2,000,000,000, at least 5,000,000,000, at least 10,000,000,000, at least20,000,000,000, at least 50,000,000,000, or at least 100,000,000,000individually addressable locations. The array may have a number ofindividually addressable locations that is within a range defined by anytwo of the preceding values. Each individually addressable location maybe digitally and/or physically accessible individually (from theplurality of individually addressable locations). For example, eachindividually addressable location may be located, identified, and/oraccessed electronically or digitally for mapping, sensing, associatingwith a device (e.g., detector, processor, dispenser, etc.), or otherwiseprocessing. Alternatively or in addition to, each individuallyaddressable location may be located, identified, and/or accessedphysically, such as for physical manipulation or extraction of ananalyte, reagent, particle, or other component located at anindividually addressable location.

Each individually addressable location may have the general shape orform of a circle, pit, bump, rectangle, or any other shape or form. Eachindividually addressable location may have a first lateral dimension(such as a radius for individually addressable locations having thegeneral shape of a circle or a width for individually addressablelocations having the general shape of a rectangle). The first lateraldimension may be at least 1 nanometer (nm), at least 2 nm, at least 5nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, atleast 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, atleast 5,000 nm, or at least 10,000 nm. The first lateral dimension maybe within a range defined by any two of the preceding values. Eachindividually addressable location may have a second lateral dimension(such as a length for individually addressable locations having thegeneral shape of a rectangle). The second lateral dimension may be atleast 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, atleast 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or atleast 10,000 nm. The second lateral dimension may be within a rangedefined by any two of the preceding values. In some instances, eachindividually addressable locations may have or be coupled to a binder,as described herein, to immobilize a analyte thereto. In some instances,only a fraction of the individually addressable locations may have or becoupled to a binder. In some instances, an individually addressablelocation may have or be coupled to a plurality of binders to immobilizean analyte thereto.

The analytes bound to the individually addressable locations mayinclude, but are not limited to, molecules, cells, organisms, nucleicacid molecules, nucleic acid colonies, beads, clusters, polonies, or DNAnanoballs. The bound analytes may be immobilized to the array in aregular, patterned, periodic, random, or pseudo-random configuration, orany other spatial arrangement.

The substrate may be configured to rotate with respect to an axis. Insome instances, the systems, devices, and apparatus described herein mayfurther comprise a rotational unit configured to rotate the substrate.The rotational unit may comprise a motor and/or a rotor to rotate thesubstrate. Such motor and/or rotor may be mechanically connected to thesubstrate directly or indirectly via intermediary components (e.g.,gears, stages, actuators, discs, pulleys, etc.). The rotational unit maybe automated. Alternatively or in addition, the rotational unit mayreceive manual input. The axis of rotation may be an axis through thecenter of the substrate. The axis may be an off-center axis. Forinstance, the substrate may be affixed to a chuck (such as a vacuumchuck) of a spin coating apparatus. The substrate may be configured torotate with a rotational velocity of at least 1 revolution per minute(rpm), at least 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm,at least 50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm,at least 1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least10,000 rpm. The substrate may be configured to rotate with a rotationalvelocity that is within a range defined by any two of the precedingvalues. The substrate may be configured to rotate with differentrotational velocities during different operations described herein. Thesubstrate may be configured to rotate with a rotational velocity thatvaries according to a time-dependent function, such as a ramp, sinusoid,pulse, or other function or combination of functions. The time-varyingfunction may be periodic or aperiodic.

A solution may be provided to the substrate prior to or during rotationof the substrate to centrifugally direct the solution across the array.In some instances, the solution may be provided to the planar arrayduring rotation of the substrate in pulses, thereby creating an annularwave of the solution moving radially outward. The pulses may haveperiodic or non-periodic (e.g., arbitrary) intervals. A series of pulsesmay comprise a series of waves producing a surface-reagent exchange. Thesurface-reagent exchange may comprise washing in which each subsequentpulse comprises a reduced concentration of the surface reagent. Thesolution may have a temperature different than that of the substrate,thereby providing a source or sink of thermal energy to the substrate orto an analyte located on the substrate. The thermal energy may provide atemperature change to the substrate or to the analyte. The temperaturechange may be transient. The temperature change may enable, disable,enhance, or inhibit a chemical reaction, such as a chemical reaction tobe carried out upon the analyte. For example, the chemical reaction maycomprise denaturation, hybridization, or annealing of nucleic acidmolecules. The chemical reaction may comprise a step in a polymerasechain reaction (PCR), bridge amplification, or other nucleic acidamplification reaction. The temperature change may modulate, increase,or decrease a signal detected from the analyte.

The array may be in fluid communication with at least one sample inlet(of a fluid channel). The array may be in fluid communication with thesample inlet via an air gap. In some cases, the array may additionallybe in fluid communication with at least one sample outlet. The array maybe in fluid communication with the sample outlet via an airgap. Thesample inlet may be configured to direct a solution to the array. Thesample outlet may be configured to receive a solution from the array.The solution may be directed to the array using one or more dispensingnozzles. For example, the solution may be directed to the array using atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 dispensing nozzles. The solutionmay be directed to the array using a number of nozzles that is within arange defined by any two of the preceding values. In some cases,different reagents (e.g., nucleotide solutions of different types,different probes, washing solutions, etc.) may be dispensed viadifferent nozzles, such as to prevent contamination. Each nozzle may beconnected to a dedicated fluidic line or fluidic valve, which mayfurther prevent contamination. A type of reagent may be dispensed viaone or more nozzles. The one or more nozzles may be directed at or inproximity to a center of the substrate. Alternatively, the one or morenozzles may be directed at or in proximity to a location on thesubstrate other than the center of the substrate. Alternatively or incombination, one or more nozzles may be directed closer to the center ofthe substrate than one or more of the other nozzles. For instance, oneor more nozzles used for dispensing washing reagents may be directedcloser to the center of the substrate than one or more nozzles used fordispensing active reagents. The one or more nozzles may be arranged atdifferent radii from the center of the substrate. Two or more nozzlesmay be operated in combination to deliver fluids to the substrate moreefficiently. One or more nozzles may be configured to deliver fluids tothe substrate as a jet, spray (or other dispersed fluid), and/ordroplets. One or more nozzles may be operated to nebulize fluids priorto delivery to the substrate.

The solution may be dispensed on the substrate while the substrate isstationary; the substrate may then be subjected to rotation followingthe dispensing of the solution. Alternatively, the substrate may besubjected to rotation prior to the dispensing of the solution; thesolution may then be dispensed on the substrate while the substrate isrotating.

Rotation of the substrate may yield a centrifugal force (or inertialforce directed away from the axis) on the solution, causing the solutionto flow radially outward over the array. In this manner, rotation of thesubstrate may direct the solution across the array. Continued rotationof the substrate over a period of time may dispense a fluid film of anearly constant thickness across the array. The rotational velocity ofthe substrate may be selected to attain a desired thickness of a film ofthe solution on the substrate. The film thickness may be related to therotational velocity by equation (1):

$\begin{matrix}{{h(t)} = \frac{\sqrt{3{\mu/2}}}{\sqrt{{2t\; {\rho\omega}^{2}} - {3\mu \; C}}}} & (1)\end{matrix}$

Here, h(t) is the thickness of the fluid film at time t, μ is theviscosity of the fluid, ω is the rotational velocity, and C is aconstant.

Alternatively or in combination, the viscosity of the solution may bechosen to attain a desired thickness of a film of the solution on thesubstrate. For instance, the rotational velocity of the substrate or theviscosity of the solution may be chosen to attain a film thickness of atleast 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), atleast 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50μm, at least 100 μm. at least 200 μm, at least 500 μm, or at least 1 mm.The rotational velocity of the substrate and/or the viscosity of thesolution may be chosen to attain a film thickness that is within a rangedefined by any two of the preceding values. The viscosity of thesolution may be controlled by controlling a temperature of the solution.The thickness of the film may be measured or monitored. Measurements ormonitoring of the thickness of the film may be incorporated into afeedback system to better control the film thickness. The thickness ofthe film may be measured or monitored by a variety of techniques. Forinstances, the thickness of the film may be measured or monitored bythin film spectroscopy with a thin film spectrometer, such as a fiberspectrometer.

The solution may be a reaction mixture comprising a variety ofcomponents. For example, the solution may comprise a plurality of probesconfigured to interact with the analyte. For example, the probes mayhave binding specificity to the analyte. In another example, the probesmay not have binding specificity to the analyte. A probe may beconfigured to permanently couple to the analyte. A probe may beconfigured to transiently couple to the analyte. For example, anucleotide probe may be permanently incorporated into a growing strandhybridized to a nucleic acid molecule analyte. Alternatively, anucleotide probe may transiently bind to the nucleic acid moleculeanalyte. Transiently coupled probes may be subsequently removed from theanalyte. Subsequent removal of the transiently coupled probes from ananalyte may or may not leave a residue (e.g., chemical residue) on theanalyte. A type of probe in the solution may depend on the type ofanalyte. A probe may comprise a functional group or moiety configured toperform specific functions. For example, a probe may comprise a label(e.g., dye). A probe may be configured to generate a detectable signal(e.g., optical signal), such as via the label, upon coupling orotherwise interacting with the analyte. In some instances, a probe maybe configured to generate a detectable signal upon activation (e.g., astimuli). In another example, a nucleotide probe may comprise reversibleterminators (e.g., blocking groups) configured to terminate polymerasereactions (until unblocked). The solution may comprise other componentsto aid, accelerate, or decelerate a reaction between the probe and theanalyte (e.g., enzymes, catalysts, buffers, saline solutions, chelatingagents, reducing agents, other agents, etc.). In some instances, thesolution may be a washing solution. In some instances, a washingsolution may be directed to the substrate to bring the washing solutionin contact with the array after a reaction or interaction betweenreagents (e.g., a probe) in a reaction mixture solution with an analyteimmobilized on the array. The washing solution may wash away any freereagents from the previous reaction mixture solution.

A detectable signal, such as an optical signal (e.g., fluorescentsignal), may be generated upon reaction between a probe in the solutionand the analyte. For example, the signal may originate from the probeand/or the analyte. The detectable signal may be indicative of areaction or interaction between the probe and the analyte. Thedetectable signal may be a non-optical signal. For example, thedetectable signal may be an electronic signal. The detectable signal maybe detected by one or more sensors. For example, an optical signal maybe detected via one or more optical detectors in an optical detectionscheme described elsewhere herein. The signal may be detected duringrotation of the substrate. The signal may be detected followingtermination of the rotation. The signal may be detected while theanalyte is in fluid contact with the solution. The signal may bedetected following washing of the solution. In some instances, after thedetection, the signal may be muted, such as by cleaving a label from theprobe and/or the analyte, and/or modifying the probe and/or the analyte.Such cleaving and/or modification may be effected by one or morestimuli, such as exposure to a chemical, an enzyme, light (e.g.,ultraviolet light), or temperature change (e.g., heat). In someinstances, the signal may otherwise become undetectable by deactivatingor changing the mode (e.g., detection wavelength) of the one or moresensors, or terminating or reversing an excitation of the signal. Insome instances, detection of a signal may comprise capturing an image orgenerating a digital output (e.g., between different images).

The operations of directing a solution to the substrate and detection ofone or more signals indicative of a reaction between a probe in thesolution and an analyte in the array may be repeated one or more times.Such operations may be repeated in an iterative manner. For example, thesame analyte immobilized to a given location in the array may interactwith multiple solutions in the multiple repetition cycles. For eachiteration, the additional signals detected may provide incremental, orfinal, data about the analyte during the processing. For example, wherethe analyte is a nucleic acid molecule and the processing is sequencing,additional signals detected at each iteration may be indicative of abase in the nucleic acid sequence of the nucleic acid molecule. Theoperations may be repeated at least 1, at least 2, at least 5, at least10, at least 20, at least 50, at least 100, at least 200, at least 500,at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 cycles to process the analyte. Insome instances, a different solution may be directed to the substratefor each cycle. For example, at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least10,000,000, at least 20,000,000, at least 50,000,000,at least 100,000,000, at least 200,000,000, at least 500,000,000, or atleast 1,000,000,000 solutions may be directed to the substrate.

In some instances, a washing solution may be directed to the substratebetween each cycle (or at least once during each cycle). For instance, awashing solution may be directed to the substrate after each type ofreaction mixture solution is directed to the substrate. The washingsolutions may be distinct. The washing solutions may be identical. Thewashing solution may be dispensed in pulses during rotation, creatingannular waves as described herein. For example, at least 1, at least 2,at least 5, at least 10, at least 20, at least 50, at least 100, atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,at least 10,000, at least 20,000, at least 50,000, at least 100,000, atleast 200,000, at least 500,000, at least 1,000,000, at least 2,000,000,at least 5,000,000, at least10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 washing solutions may be directedto the substrate.

In some instances, a subset or an entirety of the solution(s) may berecycled after the solution(s) have contacted the substrate. Recyclingmay comprise collecting, filtering, and reusing the subset or entiretyof the solution. The filtering may be molecule filtering.

Nucleic Acid Sequencing Using a Rotating Array

In some instances, a method for sequencing may employ sequencing bysynthesis schemes wherein a nucleic acid molecule is sequencedbase-by-base with primer extension reactions. For example, a method forsequencing a nucleic acid molecule may comprise providing a substratecomprising an array having immobilized thereto the nucleic acidmolecule. The array may be a planar array. The substrate may beconfigured to rotate with respect to an axis. The method may comprisedirecting a solution comprising a plurality of nucleotides across thearray prior to or during rotation of the substrate. Rotation of thesubstrate may facilitate coating of the substrate surface with thesolution. The nucleic acid molecule may be subjected to a primerextension reaction under conditions sufficient to incorporate orspecifically bind at least one nucleotide from the plurality ofnucleotides into a growing strand that is complementary to the nucleicacid molecule. A signal indicative of incorporation or binding of atleast one nucleotide may be detected, thereby sequencing the nucleicacid molecule.

In some instances, the method may comprise, prior to providing thesubstrate having immobilized thereto the nucleic acid molecule,immobilizing the nucleic acid molecule to the substrate. For example, asolution comprising a plurality of nucleic acid molecules comprising thenucleic acid molecule may be directed to the substrate prior to, during,or subsequent to rotation of the substrate, and the substrate may besubject to conditions sufficient to immobilize at least a subset of theplurality of nucleic acid molecules as an array on the substrate.

FIG. 2 shows a method 200 for sequencing a nucleic acid molecule. In afirst operation 210, the method may comprise providing a substrate, asdescribed elsewhere herein. The substrate may comprise an array of aplurality of individually addressable locations. The array may be aplanar array. The array may be a textured array. The array may be apatterned array. For example, the array may define individuallyaddressable locations with wells and/or pillars. A plurality of nucleicacid molecules, which may or may not be copies of the same nucleic acidmolecule, may be immobilized to the array. Each nucleic acid moleculefrom the plurality of nucleic acid molecules may be immobilized to thearray at a given individually addressable location of the plurality ofindividually addressable locations.

The substrate may be configured to rotate with respect to an axis. Theaxis may be an axis through the center or substantially center of thesubstrate. The axis may be an off-center axis. For instance, thesubstrate may be affixed to a chuck (such as a vacuum chuck) of a spincoating apparatus. The substrate may be configured to rotate with arotational velocity of at least 1 revolution per minute (rpm), at least2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least 10,000rpm. The substrate may be configured to rotate with a rotationalvelocity that is within a range defined by any two of the precedingvalues. The substrate may be configured to rotate with differentrotational velocities during different operations described herein. Thesubstrate may be configured to rotate with a rotational velocity thatvaries according to a time-dependent function, such as a ramp, sinusoid,pulse, or other function or combination of functions. The time-varyingfunction may be periodic or aperiodic.

In a second operation 220, the method may comprise directing a solutionacross the array prior to or during rotation of the substrate. Thesolution may be centrifugally directed across the array. In someinstances, the solution may be directed to the array during rotation ofthe substrate in pulses, thereby creating an annular wave of thesolution moving radially outward. The solution may have a temperaturedifferent than that of the substrate, thereby providing a source or sinkof thermal energy to the substrate or to a nucleic acid moleculeslocated on the substrate. The thermal energy may provide a temperaturechange to the substrate or to the nucleic acid molecule. The temperaturechange may be transient. The temperature change may enable, disable,enhance, or inhibit a chemical reaction, such as a chemical reaction tobe carried out upon the nucleic acid molecule. The chemical reaction maycomprise denaturation, hybridization, or annealing of the plurality ofnucleic acid molecules. The chemical reaction may comprise a step in apolymerase chain reaction (PCR), bridge amplification, or other nucleicacid amplification reaction. The temperature change may modulate,increase, or decrease a signal detected from the nucleic acid molecules(or from probes in the solution).

In some instances, the solution may comprise probes configured tointeract with nucleic acid molecules. For example, in some instances,such as for performing sequencing by synthesis, the solution maycomprise a plurality of nucleotides (in single bases). The plurality ofnucleotides may include nucleotide analogs, naturally occurringnucleotides, and/or non-naturally occurring nucleotides, collectivelyreferred to herein as “nucleotides.” The plurality of nucleotides may ormay not be bases of the same type (e.g., A, T, G, C, etc.). For example,the solution may or may not comprise bases of only one type. Thesolution may comprise at least 1 type of base or bases of at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 types. For instance, the solution may compriseany possible mixture of A, T, C, and G. In some instances, the solutionmay comprise a plurality of natural nucleotides and non-naturalnucleotides. The plurality of natural nucleotides and non-naturalnucleotides may or may not be bases of the same type (e.g., A, T, G, C).

One or more nucleotides of the plurality of nucleotides may beterminated (e.g., reversibly terminated). For example, a nucleotide maycomprise a reversible terminator, or a moiety that is capable ofterminating primer extension reversibly. Nucleotides comprisingreversible terminators may be accepted by polymerases and incorporatedinto growing nucleic acid sequences analogously to non-reversiblyterminated nucleotides. Following incorporation of a nucleotide analogcomprising a reversible terminator into a nucleic acid strand, thereversible terminator may be removed to permit further extension of thenucleic acid strand. A reversible terminator may comprise a blocking orcapping group that is attached to the 3′-oxygen atom of a sugar moiety(e.g., a pentose) of a nucleotide or nucleotide analog. Such moietiesare referred to as 3′-O-blocked reversible terminators. Examples of3′-O-blocked reversible terminators include, for example, 3′-ONH₂reversible terminators, 3′-O-allyl reversible terminators, and3′-O-aziomethyl reversible terminators. Alternatively, a reversibleterminator may comprise a blocking group in a linker (e.g., a cleavablelinker) and/or dye moiety of a nucleotide analog. 3′-unblockedreversible terminators may be attached to both the base of thenucleotide analog as well as a fluorescing group (e.g., label, asdescribed herein). Examples of 3′-unblocked reversible terminatorsinclude, for example, the “virtual terminator” developed by HelicosBioSciences Corp. and the “lightning terminator” developed by Michael L.Metzker et al. Cleavage of a reversible terminator may be achieved by,for example, irradiating a nucleic acid molecule including thereversible terminator.

One or more nucleotides of the plurality of nucleotides may be labeledwith a dye, fluorophore, or quantum dot. For example, the solution maycomprise labeled nucleotides. In another example, the solution maycomprise unlabeled nucleotides. In another example, the solution maycomprise a mixture of labeled and unlabeled nucleotides. Non-limitingexamples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine,Hoechst, SYBR gold, ethidium bromide, acridine, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, phenanthridines and acridines, ethidiumbromide, propidium iodide, hexidium iodide, dihydroethidium, ethidiumhomodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D,LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange,POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1,BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3,TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen,RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40,-41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11,-20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85(orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein,fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate(TRITC), rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3,Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC),Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD,ethidium homodimer I, ethidium homodimer II, ethidium homodimer III,ethidium bromide, umbelliferone, eosin, green fluorescent protein,erythrosin, coumarin, methyl coumarin, pyrene, malachite green,stilbene, lucifer yellow, cascade blue, dichlorotriazinylaminefluorescein, dansyl chloride, fluorescent lanthanide complexes such asthose including europium and terbium, carboxy tetrachloro fluorescein, 5and/or 6-carboxy fluorescein (FAM), VIC, 5-(or 6-)iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-amino-naphthalimide,phycobiliproteins, Atto 390, 425, 465, 488, 495, 532, 565, 594, 633,647, 647N, 665, 680 and 700 dyes, AlexaFluor 350, 405, 430, 488, 532,546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes,or other fluorophores, Black Hole Quencher Dyes (Biosearch Technologies)such as BH1-0, BHQ-1, BHQ-3, BHQ-10); QSY Dye fluorescent quenchers(from Molecular Probes/Invitrogen) such QSY7, QSY9, QSY21, QSY35, andother quenchers such as Dabcyl and Dabsyl; CySQ and Cy7Q and DarkCyanine dyes (GE Healthcare); Dy-Quenchers (Dyomics), such as DYQ-660and DYQ-661; and ATTO fluorescent quenchers (ATTO-TEC GmbH), such asATTO 540Q, 580Q, 612Q. In some cases, the label may be one with linkers.For instance, a label may have a disulfide linker attached to the label.Non-limiting examples of such labels include Cy5-azide, Cy-2-azide,Cy-3-azide, Cy-3.5-azide, Cy5.5-azide and Cy-7-azide. In some cases, alinker may be a cleavable linker. In some cases, the label may be a typethat does not self-quench or exhibit proximity quenching. Non-limitingexamples of a label type that does not self-quench or exhibit proximityquenching include Bimane derivatives such as Monobromobimane.Alternatively, the label may be a type that self-quenches or exhibitsproximity quenching. Non-limiting examples of such labels includeCy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide andCy-7-azide. In some instances, a blocking group of a reversibleterminator may comprise the dye.

The solution may be directed to the array using one or more nozzles. Insome cases, different reagents (e.g., nucleotide solutions of differenttypes, washing solutions, etc.) may be dispensed via different nozzles,such as to prevent contamination. Each nozzle may be connected to adedicated fluidic line or fluidic valve, which may further preventcontamination. A type of reagent may be dispensed via one or morenozzles. The one or more nozzles may be directed at or in proximity to acenter of the substrate. Alternatively, the one or more nozzles may bedirected at or in proximity to a location on the substrate other thanthe center of the substrate. Two or more nozzles may be operated incombination to deliver fluids to the substrate more efficiently.

The solution may be dispensed on the substrate while the substrate isstationary; the substrate may then be subjected to rotation followingthe dispensing of the solution. Alternatively, the substrate may besubjected to rotation prior to the dispensing of the solution; thesolution may then be dispensed on the substrate while the substrate isrotating. Rotation of the substrate may yield a centrifugal force (orinertial force directed away from the axis) on the solution, causing thesolution to flow radially outward over the array.

In a third operation 230, the method may comprise subjecting the nucleicacid molecule to a primer extension reaction. The primer extensionreaction may be conducted under conditions sufficient to incorporate atleast one nucleotide from the plurality of nucleotides into a growingstrand that is complementary to the nucleic acid molecule. Thenucleotide incorporated may or may not be labeled.

In some cases, the operation 230 may further comprise modifying at leastone nucleotide. Modifying the nucleotide may comprise labeling thenucleotide. For instance, the nucleotide may be labeled, such as with adye, fluorophore, or quantum dot. The nucleotide may be cleavablylabeled. In some instances, modifying the nucleotide may compriseactivating (e.g., stimulating) a label of the nucleotide.

In a fourth operation 240, the method may comprise detecting a signalindicative of incorporation of the at least one nucleotide. The signalmay be an optical signal. The signal may be a fluorescence signal. Thesignal may be detected during rotation of the substrate. The signal maybe detected following termination of the rotation. The signal may bedetected while the nucleic acid molecule to be sequenced is in fluidcontact with the solution. The signal may be detected following fluidcontact of the nucleic acid molecule with the solution. The operation240 may further comprise modifying a label of the at least onenucleotide. For instance, the operation 240 may further comprisecleaving the label of the nucleotide (e.g., after detection). Thenucleotide may be cleaved by one or more stimuli, such as exposure to achemical, an enzyme, light (e.g., ultraviolet light), or heat. Once thelabel is cleaved, a signal indicative of the incorporated nucleotide maynot be detectable with one or more detectors.

The method 200 may further comprise repeating operations 220, 230,and/or 240 one or more times to identify one or more additional signalsindicative of incorporation of one or more additional nucleotides,thereby sequencing the nucleic acid molecule. The method 200 maycomprise repeating operations 220, 230, and/or 240 in an iterativemanner. For each iteration, an additional signal may indicateincorporation of an additional nucleotide. The additional nucleotide maybe the same nucleotide as detected in the previous iteration. Theadditional nucleotide may be a different nucleotide from the nucleotidedetected in the previous iteration. In some instances, at least onenucleotide may be modified (e.g., labeled and/or cleaved) between eachiteration of the operations 220, 230, or 240. For instance, the methodmay comprise repeating the operations 220, 230, and/or 240 at least 1,at least 2, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, at least 10,000, at least 20,000, at least 50,000, at least100,000, at least 200,000, at least 500,000, at least 1,000,000, atleast 2,000,000, at least 5,000,000, at least 10,000,000, at least20,000,000, at least 50,000,000, at least 100,000,000, at least200,000,000, at least 500,000,000, or at least 1,000,000,000 times. Themethod may comprise repeating the operations 220, 230, and/or 240 anumber of times that is within a range defined by any two of thepreceding values. The method 200 may thus result in the sequencing of anucleic acid molecule of any size.

The method may comprise directing different solutions to the arrayduring rotation of the substrate in a cyclical manner. For instance, themethod may comprise directing a first solution containing a first typeof nucleotide (e.g., in a plurality of nucleotides of the first type) tothe array, followed by a second solution containing a second type ofnucleotide, followed by a third type of nucleotide, followed by a fourthtype of nucleotide, etc. In another example, different solutions maycomprise different combinations of types of nucleotides. For example, afirst solution may comprise a first canonical type of nucleotide (e.g.,A) and a second canonical type of nucleotide (e.g., C), and a secondsolution may comprise the first canonical type of nucleotide (e.g., A)and a third canonical type of nucleotide (e.g., T), and a third solutionmay comprise the first canonical type, second canonical type, thirdcanonical type, and a fourth canonical type (e.g., G) of nucleotide. Inanother example, a first solution may comprise labeled nucleotides, anda second solution may comprise unlabeled nucleotides, and a thirdsolution may comprise a mixture of labeled and unlabeled nucleotides.The method may comprise directing at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least10,000,000, at least 20,000,000, at least 50,000,000,at least 100,000,000, at least 200,000,000, at least 500,000,000, or atleast 1,000,000,000 solutions to the array. The method may comprisedirecting a number of solutions that is within a range defined by anytwo of the preceding values to the array. The solutions may be distinct.The solutions may be identical.

The method may comprise directing at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least10,000,000, at least 20,000,000, at least 50,000,000,at least 100,000,000, at least 200,000,000, at least 500,000,000, or atleast 1,000,000,000 washing solutions to the substrate. For instance, awashing solution may be directed to the substrate after each type ofnucleotide is directed to the substrate. The washing solutions may bedistinct. The washing solutions may be identical. The washing solutionmay be dispensed in pulses during rotation, creating annular waves asdescribed herein.

The method may further comprise recycling a subset or an entirety of thesolution(s) after the solution(s) have contacted the substrate.Recycling may comprise collecting, filtering, and reusing the subset orentirety of the solution. The filtering may be molecule filtering.

The operations 220 and 230 may occur at a first location and theoperation 240 may occur at a second location. The first and secondlocations may comprise first and second processing bays, respectively,as described herein (for instance, with respect to FIG. 12G). The firstand second locations may comprise first and second rotating spindles,respectively, as described herein (for instance, with respect to FIG.13). The first rotating spindle may be exterior or interior to thesecond rotating spindle. The first and second rotating spindles may beconfigured to rotate with different angular velocities. Alternatively,the operation 220 may occur at a first location and the operations 230and 240 may occur at the second location.

The method may further comprise transferring the substrate between thefirst and second locations. Operations 220 and 230 may occur while thesubstrate is rotated at a first angular velocity and operation 240 mayoccur while the substrate is rotated at a second angular velocity. Thefirst angular velocity may be less than the second angular velocity. Thefirst angular velocity may be between about 0 rpm and about 100 rpm. Thesecond angular velocity may be between about 100 rpm and about 1,000rpm. Alternatively, the operation 220 may occur while the substrate isrotated at the first angular velocity and the operations 230 and 240 mayoccur while the substrate is rotated at the second angular velocity.

Many variations, alterations, and adaptations based on the method 200provided herein are possible. For example, the order of the operationsof the method 200 may be changed, some of the operations removed, someof the operations duplicated, and additional operations added asappropriate. Some of the operations may be performed in succession. Someof the operations may be performed in parallel. Some of the operationsmay be performed once. Some of the operations may be performed more thanonce. Some of the operations may comprise sub-operations. Some of theoperations may be automated. Some of the operations may be manual.

For example, in some cases, in the third operation 230, instead offacilitating a primer extension reaction, the nucleic acid molecule maybe subject to conditions to allow transient binding of a nucleotide fromthe plurality of nucleotides to the nucleic acid molecule. Thetransiently bound nucleotide may be labeled. The transiently boundnucleotide may be removed, such as after detection (e.g., see operation240). Then, a second solution may be directed to the substrate, thistime under conditions to facilitate the primer extension reaction, suchthat a nucleotide of the second solution is incorporated (e.g., into agrowing strand hybridized to the nucleic acid molecule). Theincorporated nucleotide may be unlabeled. After washing, and withoutdetecting, another solution of labeled nucleotides may be directed tothe substrate, such as for another cycle of transient binding.

In some instances, such as for performing sequencing by ligation, thesolution may comprise different probes. For example, the solution maycomprise a plurality of oligonucleotide molecules. For example, theoligonucleotide molecules may have a length of about 2 bases, 3 bases, 4bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases or more.The oligonucleotide molecules may be labeled with a dye (e.g.,fluorescent dye), as described elsewhere herein. In some instances, suchas for detecting repeated sequences in nucleic acid molecules, such ashomopolymer repeated sequences, dinucleotide repeated sequences, andtrinucleotide repeated sequences, the solution may comprise targetedprobes (e.g., homopolymer probe) configured to bind to the repeatedsequences. The solution may comprise one type of probe (e.g.,nucleotides). The solution may comprise different types of probes (e.g.,nucleotides, oligonucleotide molecules, etc.). The solution may comprisedifferent types of probes (e.g., oligonucleotide molecules, antibodies,etc.) for interacting with different types of analytes (e.g., nucleicacid molecules, proteins, etc.). Different solutions comprisingdifferent types of probes may be directed to the substrate any number oftimes, with or without detection between consecutive cycles (e.g.,detection may be performed between some consecutive cycles, but notbetween some others), to sequence or otherwise process the nucleic acidmolecule, depending on the type of processing.

FIG. 3 shows a system 300 for sequencing a nucleic acid molecule orprocessing an analyte. The system may be configured to implement themethod 200 or 1400. Although the systems (e.g., 300, 400, 500 a, 500 b,etc.) are described with respect to processing nucleic acid molecules,the systems may be used to process any other type of biological analyte,as described herein.

The system may comprise a substrate 310. The substrate may comprise anysubstrate described herein, such as any substrate described herein withrespect to FIG. 2. The substrate may comprise an array. The substratemay be open. The array may comprise one or more locations 320 configuredto immobilize one or more nucleic acid molecules or analytes. The arraymay comprise any array described herein, such as any array describedherein with respect to method 200. For instance, the array may comprisea plurality of individually addressable locations. The array maycomprise a linker (e.g., any binder described herein) that is coupled tothe nucleic acid molecule to be sequenced. Alternatively or incombination, the nucleic acid molecule to be sequenced may be coupled toa bead; the bead may be immobilized to the array. The array may betextured. The array may be a patterned array. The array may be planar.

The substrate may be configured to rotate with respect to an axis 305.The axis may be an axis through the center of the substrate. The axismay be an off-center axis. The substrate may be configured to rotate atany rotational velocity described herein, such as any rotationalvelocity described herein with respect to method 200 or 1400.

The substrate may be configured to undergo a change in relative positionwith respect to first or second longitudinal axes 315 and 325. Forinstance, the substrate may be translatable along the first and/orsecond longitudinal axes (as shown in FIG. 3). Alternatively, thesubstrate may be stationary along the first and/or second longitudinalaxes. Alternatively or in combination, the substrate may be translatablealong the axis (as shown in FIG. 4). Alternatively or in combination,the substrate may be stationary along the axis. The relative position ofthe substrate may be configured to alternate between positions. Therelative position of the substrate may be configured to alternatebetween positions with respect to one or more of the longitudinal axesor the axis. The relative position of the substrate may be configured toalternate between positions with respect to any of the fluid channelsdescribed herein. For instance, the relative position of the substratemay be configured to alternate between a first position and a secondposition. The relative position of the substrate may be configured toalternate between at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 positions. Therelative position of the substrate may be configured to alternatebetween a number of positions that is within a range defined by any twoof the preceding values. The first or second longitudinal axes may besubstantially perpendicular with the axis. The first or secondlongitudinal axes may be substantially parallel with the axis. The firstor second longitudinal axes may be coincident with the axis.

The system may comprise a first fluid channel 330. The first fluidchannel may comprise a first fluid outlet port 335. The first fluidoutlet port may be configured to dispense a first fluid to the array.The first fluid outlet port may be configured to dispense any fluiddescribed herein, such as any solution described herein. The first fluidoutlet port may be external to the substrate. The first fluid outletport may not contact the substrate. The first fluid outlet port may be anozzle. The first fluid outlet port may have an axis that issubstantially coincident with the axis. The first fluid outlet port mayhave an axis that is substantially parallel to the axis.

The system may comprise a second fluid channel 340. The second fluidchannel may comprise a second fluid outlet port 345. The second fluidoutlet port may be configured to dispense a second fluid to the array.The second fluid outlet port may be configured to dispense any fluiddescribed herein, such as any solution described herein. The secondfluid outlet port may be external to the substrate. The second fluidoutlet port may not contact the substrate. The second fluid outlet portmay be a nozzle. The second fluid outlet port may have an axis that issubstantially coincident with the axis. The second fluid outlet port mayhave an axis that is substantially parallel to the axis.

The first and second fluids may comprise different types of reagents.For instance, the first fluid may comprise a first type of nucleotide,such as any nucleotide described herein, or a nucleotide mixture. Thesecond fluid may comprise a second type of nucleotide, such as anynucleotide described herein, or a nucleotide mixture. Alternatively, thefirst and second fluids may comprise the same type of reagents (e.g.,same type of fluid is dispensed through multiple fluid outlet ports(e.g., nozzles) to increase coating speed). Alternatively or incombination, the first or second fluid may comprise a washing reagent.The first fluid channel 330 and the second fluid channel 340 may befluidically isolated. Beneficially, where the first and second fluidscomprise different types of reagents, each of the different reagents mayremain free of contamination from the other reagents during dispensing.

The first fluid outlet port may be configured to dispense the firstfluid during rotation of the substrate. The second fluid outlet port maybe configured to dispense the second fluid during rotation of thesubstrate. The first and second fluid outlet ports may be configured todispense at non-overlapping times. Alternatively, the first and secondfluid outlet ports may be configured to dispense at overlapping times,such as when the first fluid and the second fluid comprise the same typeof reagents. The substrate may be configured to rotate with a differentspeed or a different number of rotations when the first and secondoutlet ports dispense. Alternatively, the substrate may be configured torotate with the same speed and number of rotations when the first andsecond outlet ports dispense. During rotation, the array may beconfigured to direct the first fluid in a substantially radial directionaway from the axis. The first fluid outlet port may be configured todirect the first fluid to the array during at least 1, at least 2, atleast 5, at least 10, at least 20, at least 50, at least 100, at least200, at least 500, at least 1,000, at least 2,000, at least 5,000, atleast 10,000, at least 20,000, at least 50,000, at least 100,000, atleast 200,000, at least 500,000, or at least 1,000,000 full rotations ofthe substrate. The first fluid outlet port may be configured to directthe first fluid to the array during a number of full rotations that iswithin a range defined by any two of the preceding values.

The system may comprise a third fluid channel 350 comprising a thirdfluid outlet port 355 configured to dispense a third fluid. The systemmay comprise a fourth fluid channel 360 comprising a fourth fluid outletport 365 configured to dispense a fourth fluid. The third and fourthfluid channels may be similar to the first and second fluid channelsdescribed herein. The third and fourth fluids may be the same ordifferent fluids as the first and/or second fluids. In some cases, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more fluids (or reagents)may be employed. For example, 5-10 fluids (or reagents) may be employed.

Although FIG. 3 shows a change in position of the substrate, as analternative or in addition to, one or more of the first, second, third,and fourth fluid channels may be configured to undergo a change inposition. For instance, any of the first, second, third, or fourth fluidchannel may be translatable along the first and/or second longitudinalaxes. Alternatively, any of the first, second, third, or fourth fluidchannel may be stationary along the first and/or second longitudinalaxes. Alternatively or in addition to, any of the first, second, third,or fourth fluid channel may be translatable along the axis.Alternatively or in addition to, any of the first, second, third, orfourth fluid channel may be stationary along the axis.

The relative position of one or more of the first, second, third, andfourth fluid channels may be configured to alternate between positionswith respect to one or more of the longitudinal axes or the axis. Forinstance, the relative position of any of the first, second, third, orfourth fluid channel may be configured to alternate between a firstposition and a second position (e.g., by moving such channel, by movingthe substrate, or by moving the channel and the substrate). The relativeposition of any of the first, second, third, or fourth fluid channel maybe configured to alternate between at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20 or more positions. The relative position of any of the first, second,third, or fourth fluid channel may be configured to alternate between anumber of positions that is within a range defined by any two of thepreceding values. The first or second longitudinal axes may besubstantially perpendicular to the axis. The first or secondlongitudinal axes may be substantially parallel to the axis. The firstor second longitudinal axes may be coincident with the axis.

In some instances, the system may comprise one or more fluid channelsfor receiving fluid from the substrate (not shown in FIG. 3). Referringto FIG. 4A-4B, a fifth fluid channel 430 may comprise a first fluidinlet port 435. The first fluid inlet port may be located at a firstlevel of the axis (as shown in FIG. 4). In some instances, the firstfluid inlet port may surround the periphery of the substrate 310 (e.g.,circularly). The first fluid inlet port may be downstream of and influid communication with the substrate 310 when the substrate is in afirst position, such as with respect to the axis. The fifth fluidchannel may be in fluid communication with the first fluid channel 330.For example, the first fluid inlet port may be configured to receive asolution passing from the first fluid outlet port to the substrate andthereafter off the substrate (e.g., due to inertial forces duringrotation of the substrate). For instance, the first fluid inlet port maybe configured to receive the solution in a recycling process such as therecycling process described herein with respect to method 200 or 1400.In some instances, the solution received by the fifth fluid channel viathe first fluid inlet port may be fed back (e.g., after filtering) tothe first fluid channel to be dispensed via the first fluid outlet portto the substrate. The fifth fluid channel and the first fluid channelmay define at least part of a first cyclic fluid flow path. The firstcyclic fluid flow path may comprise a filter, such as a filter describedherein with respect to method 200 or 1400. The filter may be a molecularfilter. In other instances, the solution received by the fifth fluidchannel may be fed back (e.g., after filtering) to different fluidchannels (other than the first fluid channel) to be dispensed viadifferent fluid outlet ports.

The system may comprise a sixth fluid channel 440. The sixth fluidchannel may comprise a second fluid inlet port 445. The second fluidinlet port may be located at a second level of the axis (as shown inFIG. 4). In some instances, the second fluid inlet port may surround theperiphery of the substrate 310. The second fluid inlet port may bedownstream of and in fluid communication with the substrate 310 when thesubstrate is in a second position, such as with respect to the axis. Thesixth fluid channel may be in fluid communication with the second fluidchannel 340. For example, the second fluid inlet port may be configuredto receive a solution passing from the second fluid outlet port to thesubstrate and thereafter off the substrate. For instance, the secondfluid inlet port may be configured to receive the solution in arecycling process such as the recycling process described herein withrespect to method 200 or 1400. In some instances, the solution receivedby the sixth fluid channel via the second fluid inlet port may be fedback (e.g., after filtering) to the second fluid channel to be dispensedvia the second fluid outlet port to the substrate. The sixth fluidchannel and the second fluid channel may define at least part of asecond cyclic fluid flow path. The second cyclic fluid flow path maycomprise a filter, such as a filter described herein with respect tomethod 200 or 1400. The filter may be a molecular filter.

The system may comprise a shield (not shown) that prevents fluidcommunication between the substrate and the second fluid inlet port whenthe substrate is in the first position and between the substrate and thefirst fluid inlet port when the substrate is in the second position.

The system may further comprise one or more detectors 370. The detectorsmay be optical detectors, such as one or more photodetectors, one ormore photodiodes, one or more avalanche photodiodes, one or morephotomultipliers, one or more photodiode arrays, one or more avalanchephotodiode arrays, one or more cameras, one or more charged coupleddevice (CCD) cameras, or one or more complementary metal oxidesemiconductor (CMOS) cameras. The cameras may be TDI or other continuousarea scanning detectors described herein. The detectors may befluorescence detectors. The detectors may be in sensing communicationwith the array. For instance, the detectors may be configured to detecta signal from the array. The signal may be an optical signal. The signalmay be a fluorescence signal. The detectors may be configured to detectthe signal from the substrate during rotation of the substrate. Thedetectors may be configured to detect the signal from the substrate whenthe substrate is not rotating. The detectors may be configured to detectthe signal from the substrate following termination of the rotation ofthe substrate. FIG. 3 shows an example region 375 on the substrate thatis optically mapped to the detector.

The system may comprise one or more sources (not shown in FIG. 3)configured to deliver electromagnetic radiation to the substrate. Thesources may comprise one or more optical sources. The sources maycomprise one or more incoherent or coherent optical sources. The sourcesmay comprise one or more narrow bandwidth or broadband optical sources.The sources may be configured to emit optical radiation having abandwidth of at most 1 hertz (Hz), at most 2 Hz, at most 5 Hz, at most10 Hz, at most 20 Hz, at most 50 Hz, at most 100 Hz, at most 200 Hz, atmost 500 Hz, at most 1 kilohertz (kHz), at most 2 kHz, at most 5 kHz, atmost 10 kHz, at most 20 kHz, at most 50 kHz, at most 100 kHz, at most200 kHz, at most 500 kHz, at most 1 megahertz (MHz), at most 2 MHz, atmost 5 MHz, at most 10 MHz, at most 20 MHz, at most 50 MHz, at most 100MHz, at most 200 MHz, at most 500 MHz, at most 1 gigahertz (GHz), atmost 2 GHz, at most 5 GHz, at most 10 GHz, at most 20 GHz, at most 50GHz, at most 100 GHz, or a bandwidth that is within a range defined byany two of the preceding values. The sources may comprise one or morelasers. The sources may comprise one or more single-mode laser sources.The sources may comprise one or more multi-mode laser sources. Thesources may comprise one or more laser diodes. The source may compriseone or more light emitting diodes (LEDs). The sources may be configuredto emit light comprising one or more wavelengths in the ultraviolet(about 100 nm to about 400 nm), visible (about 400 nm to about 700 nm),or infrared (about 700 nm to about 10,000 nm) regions of theelectromagnetic spectrum, or any combination therefore. For instances,the sources may emit radiation comprising one or more wavelengths in therange from 600 nm to 700 nm. The sources may emit radiation, eitherindividually or in combination, having an optical power of at least 0.05watts (W), at least 0.1 W, at least 0.2 W, at least 0.5 W, at least 1 W,at least 2 W, at least 5 W, at least 10 W, or an optical power that iswithin a range defined by any two of the preceding values. The sourcesmay be configured to interact with molecules on the substrate togenerate detectable optical signals that may be detected by the opticaldetectors. For instance, the sources may be configured to generateoptical absorption, optical reflectance, scattering, phosphorescence,fluorescence, or any other optical signal described herein.

The system may comprise a seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth,eighteenth, nineteenth, or twentieth fluid channel. Each fluid channelmay comprise a fluid outlet port or a fluid inlet port in fluidcommunication with the substrate. For instance, the ninth, tenth,thirteenth, fourteenth, seventeenth, or eighteenth fluid channel maycomprise a fluid outlet port. The seventh, eighth, eleventh, twelfth,fifteenth, sixteenth, nineteenth, or twentieth fluid channel maycomprise a fluid inlet port. Alternatively, the system may comprise morethan twenty fluid channels comprising a fluid outlet port or a fluidinlet port.

Thus, the system may comprise fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be configured to dispense fifth, sixth,seventh, eighth, ninth, or tenth fluids to the array. The fifth, sixth,seventh, eighth, ninth, or tenth fluid outlet ports may be configured todispense any fluid described herein, such as any solution describedherein. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outletports may be similar to the first, second, third, or fourth fluid outletports described herein. Alternatively, the system may comprise more thanten fluid outlet ports.

The fluid channels may be fluidically isolated from one another. Forinstance, the fluid channels may be fluidically isolated upstream of thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be external to the substrate. The fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports may notcontact the substrate. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be a nozzle.

The system may comprise third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth fluid inlet ports. The third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports may be in fluidcommunication with the substrate when the substrate is in a third,fourth, fifth, sixth, seventh, eighth, ninth, or tenth position (e.g.,with respect to the axis), respectively. Alternatively, the system maycomprise more than ten fluid inlet ports.

The ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenthfluid channel may be in fluid communication with the seventh, eighth,eleventh, twelfth, fifteenth, or sixteenth, fluid channel, respectively;each pair of fluid channels may define at least part of a third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth cyclic fluid flow path,respectively. Each cyclic fluid flow path may be configured similarly tothe first or second cyclic fluid flow paths described herein, with thefluid inlet port of the cyclic fluid flow path configured to receive asolution passing from the fluid outlet port of the cyclic fluid flowpath to the substrate. Each cyclic fluid flow path may be configured toreceive the solution in a recycling process as described herein. Eachcyclic fluid flow path may comprise a filter as described herein.

The fifth, sixth, seventh, eighth, ninth, or tenth fluids may comprisedifferent types of reagents. For instance, the fifth, sixth, seventh,eighth, ninth, or tenth fluid may comprise a fifth, sixth, seventh,eighth, ninth, or tenth type of nucleotide, respectively, such as anynucleotide described herein. Alternatively or in combination, the fifth,sixth, seventh, eighth, ninth, or tenth fluid may comprise a washingreagent.

The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port maybe configured to dispense the fifth, sixth, seventh, eighth, ninth, ortenth fluid, respectively, during rotation of the substrate. The fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports may beconfigured to dispense at overlapping or non-overlapping times.

FIG. 4A shows a system 400 for sequencing a nucleic acid molecule in afirst vertical level. The system may be substantially similar to system300 described herein or may differ from system 300 in the arrangement ofone or more of its elements. The system 400 may comprise substrate 310described herein. The system 400 may utilize vertical motion parallel tothe axis 305 to expose (e.g., make available fluid communication) thesubstrate 310 to different fluid channels. The system may comprise firstfluid channel 330 and first fluid outlet port 335 described herein. Thesystem may comprise second fluid channel 340 and second fluid outletport 345 described herein. The system may comprise third fluid channel350 and third fluid outlet port 355 described herein. The system maycomprise fourth fluid channel 360 and fourth fluid outlet port 365described herein. The system may comprise detector 370 described herein.The detector may be in optical communication with the region shown. Thesystem may comprise any optical source described herein (not shown inFIG. 4A).

The fifth fluid channel 430 and first fluid inlet port 435 may bearranged at a first level along the vertical axis, as shown in FIGS. 4Aand 4B. The sixth fluid channel 440 and second fluid inlet port 445 maybe arranged at a second level along the vertical axis. In this manner,the system may be viewed as comprising first and second fluid flowpaths, with each fluid flow path located at a different vertical level.The substrate 310 may be vertically movable between the first level andthe second level, from the first level to the second level, and from thesecond level to the first level. As an alternative, the substrate may bevertically fixed but the levels may be vertically movable with respectto the substrate 310. As another alternative, the substrate and thelevels may be vertically movable.

The system 400 may comprise multiple levels. The levels may bevertically orientated relative to one another. The system may include atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more levels.Each level may include one or more sub-levels (e.g., an incrementallevel between any two levels). Each level may be for dispensing and/orrecovering a different fluid (or reagent). Some levels may be fordispensing the same fluid (or reagent).

While in the first vertical level, the substrate may be in fluidcommunication with the fifth fluid channel and the first fluid inletport, but not the sixth fluid channel and the second fluid inlet port.The substrate may be isolated from the sixth fluid channel and thesecond fluid inlet port by a shield (not shown), as described herein. Afirst fluid or first solution described herein may be dispensed to thesubstrate while the substrate is in this first vertical level. Forexample, any excess of the first solution spinning off the substrate maybe received by the first fluid inlet port while the substrate is at thefirst vertical level. In another example, a washing solution (e.g.,dispensed from a different fluid outlet port than the first fluid)spinning off the substrate with some of the first fluid may be receivedby the first fluid inlet port while the substrate is at the firstvertical level. The substrate may then be moved to a second verticallevel by vertically moving the substrate. Alternatively, the fifth orsixth fluid channels may be moved vertically. Alternatively or inaddition, the substrate and one or more of the fluid channels may bemoved relative to the other (e.g., along the axis).

FIG. 4B shows the system 400 for sequencing a nucleic acid molecule in asecond vertical level. While in the second vertical level, the substratemay be in fluid communication with the sixth fluid channel and thesecond fluid inlet port, but not the fifth fluid channel and the firstfluid inlet port. The substrate may be isolated from the fifth fluidchannel and the first fluid inlet port by a shield (not shown), asdescribed herein. A second fluid or second solution described herein maybe dispensed to the substrate while the substrate is in this secondvertical position. Alternatively, the first solution may be removedwhile the substrate is in the second vertical position. In some cases,the first solution may be recycled while the substrate is in the secondvertical position. The substrate may then be moved back to the firstvertical level, or to another vertical level described herein, byvertically moving the substrate. Alternatively, the fifth or sixth fluidchannels may be moved vertically. Alternatively or in addition, thesubstrate and one or more of the fluid channels may be moved relative tothe other (e.g., along the axis).

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidinlet ports may be located at third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth vertical levels, respectively. The substrate maybe moved to the third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth vertical levels by vertically moving the substrate or byvertically moving the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth,or twentieth fluid flow channels. At any of the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth or more verticallevels, any fluid solution described herein may be dispensed to thesubstrate. At any of the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth or more vertical levels, any fluidsolution described herein may be removed from the substrate. At any ofthe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth or more vertical levels, any fluid solution described herein maybe recycled from the substrate.

FIG. 5A shows a first example of a system 500 a for sequencing a nucleicacid molecule using an array of fluid flow channels. The system may besubstantially similar to system 300 or 400 described herein and maydiffer from system 300 or 400 in the arrangement of one or more of itselements. The system 500 a may utilize a geometrical arrangement of aplurality of fluid flow channels to expose the substrate to differentfluids. The system 500 a may comprise substrate 310 described herein.The system may comprise first fluid channel 330 and first fluid outletport 335 described herein. The system may comprise second fluid channel340 and second fluid outlet port 345 described herein. The system maycomprise fifth fluid channel 430 and first fluid inlet port 435described herein (not shown in FIG. 5A). The system may comprise sixthfluid channel 440 and second fluid inlet port 445 described herein (notshown in FIG. 5A). The system may comprise detector 370 described herein(not shown in FIG. 5A). The system may comprise any optical sourcedescribed herein (not shown in FIG. 5A).

The first fluid channel and first fluid outlet port may be arranged at afirst position, as shown in FIG. 5A. The second fluid channel and secondfluid outlet port may be arranged at a second position. The system maybe configured to dispense a first fluid from the first fluid outlet portand a second fluid from the second fluid outlet port.

The system may comprise any of third, fourth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, or twentieth fluid channelsdescribed herein. The system may comprise any of third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports describedherein. The system may comprise any of third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidoutlet ports may be located at third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth positions, respectively. The system may beconfigured to dispense a third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth fluid from the third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth fluid outlet port, respectively.

Any two or more of the first, second, third, fourth, seventh, eighth,ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth,sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or more fluidchannels may form an array of fluid flow channels. The array of fluidflow channels may be moveable. Alternatively, the array of fluid flowchannels may be at a fixed location with respect to the substrate. Eachfluid flow channel of the array of fluid flow channels may be positionedsuch that a longitudinal axis of the fluid flow channel forms an anglewith the rotational axis of the substrate. The angle may have a value ofat least 0 degrees, at least 5 degrees, at least 10 degrees, at least 15degrees, at least 20 degrees, at least 25 degrees, at least 30 degrees,at least 35 degrees, at least 40 degrees, at least 45 degrees, at least50 degrees, at least 55 degrees, at least 60 degrees, at least 65degrees, at least 70 degrees, at least 75 degrees, at least 80 degrees,at least 85 degrees, or at least 90 degrees. The angle may have a valuethat is within a range defined by any two of the preceding values. Eachfluid channel of the array of fluid channels may make a similar anglewith the substrate. Alternatively, one or more fluid channels may makedifferent angles with the substrate.

FIG. 5B shows a second example of a system 500 b for sequencing anucleic acid molecule using an array of fluid flow channels.

The system may be substantially similar to system 300 or 400 describedherein and may differ from system 300 or 400 in the arrangement of oneor more of its elements. The system 500 b may utilize a plurality offluid flow channels configured to move relative to the substrate toexpose the substrate to different fluids. The system 500 b may comprisesubstrate 310 described herein. The system may comprise first fluidchannel 330 and first fluid outlet port 335 described herein. The systemmay comprise second fluid channel 340 and second fluid outlet port 345described herein. The system may comprise fifth fluid channel 430 andfirst fluid inlet port 435 described herein (not shown in FIG. 5B). Thesystem may comprise sixth fluid channel 440 and second fluid inlet port445 described herein (not shown in FIG. 5B). The system may comprisedetector 370 described herein (not shown in FIG. 5B). The system maycomprise any optical source described herein (not shown in FIG. 5B).

The first fluid channel and first fluid outlet port may be attached to afluid dispenser 510. The fluid dispenser may be a moveable fluiddispenser, such as comprising a moveable gantry arm, as shown in FIG.5B. As an alternative, the fluid dispenser may be fixed or stationary.The fluid dispenser may be configured to move to a first position todispense a first fluid from the first fluid outlet port. The secondfluid channel and second fluid outlet port may also be attached to thefluid dispenser. The fluid dispenser may be configured to move to asecond position to dispense a second fluid from the second fluid outletport.

The system may comprise any of third, fourth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, or twentieth fluid channelsdescribed herein. The system may comprise any of third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports describedherein. The system may comprise any of third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidoutlet ports may be attached to the fluid dispenser. The fluid dispensermay be configured to move to a third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth position to dispense a third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid from the third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port,respectively. Alternatively, the fluid dispenser may be kept stationaryand the substrate 310 may be moved to different positions to receivedifferent fluids.

FIG. 6 shows a computerized system 600 for sequencing a nucleic acidmolecule. The system may comprise a substrate 310, such as a substratedescribed herein with respect to method 200 or 1400, or system 300. Thesystem may further comprise a fluid flow unit 610. The fluid flow unitmay comprise any element associated with fluid flow described herein,such as any or all of elements 330, 335, 340, 345, 350, 355, 360, 365,430, 435, 440, 445, and 370 described herein with respect to system 300,400, 500 a, or 500 b. The fluid flow unit may be configured to direct asolution comprising a plurality of nucleotides described herein to anarray of the substrate prior to or during rotation of the substrate. Thefluid flow unit may be configured to direct a washing solution describedherein to an array of the substrate prior to or during rotation of thesubstrate. In some instances, the fluid flow unit may comprise pumps,compressors, and/or actuators to direct fluid flow from a first locationto a second location. With respect to method 1400, the fluid flow systemmay be configured to direct any solution to the substrate 310. Withrespect to method 1400, the fluid flow system may be configured tocollect any solution from the substrate 310. The system may furthercomprise a detector 370, such as any detector described herein withrespect to system 300 or 400. The detector may be in sensingcommunication with the array of the substrate.

The system may further comprise one or more computer processors 620. Theone or more processors may be individually or collectively programmed toimplement any of the methods described herein. For instance, the one ormore processors may be individually or collectively programmed toimplement any or all operations of the methods of the presentdisclosure, such as method 200 or 1400. In particular, the one or moreprocessors may be individually or collectively programmed to: (i) directthe fluid flow unit to direct the solution comprising the plurality ofnucleotides across the array during or prior to rotation of thesubstrate; (ii) subject the nucleic acid molecule to a primer extensionreaction under conditions sufficient to incorporate at least onenucleotide from the plurality of nucleotides into a growing strand thatis complementary to the nucleic acid molecule; and (iii) use thedetector to detect a signal indicative of incorporation of the at leastone nucleotide, thereby sequencing the nucleic acid molecule.

While the rotational system has been described with respect tosequencing applications, such rotational schemes may be used for otherapplications (e.g., pre-sequencing applications, sample preparation,etc.), such as template seeding and surface amplification processes. Forexample, the reagents dispensed during or prior to rotation of thesubstrate may be tailored to the other applications. While the reagentsdispensed to the substrate in the rotational system have been describedwith respect to nucleotides, any reagent that may react with a nucleicacid molecule (or any other molecule or cell) immobilized to thesubstrate, such as probes, adaptors, enzymes, and labelling reagents,may be dispensed to the substrate prior to, during, or subsequent torotation to achieve high speed coating of the substrate with thedispensed reagents.

The systems for sequencing nucleic acid molecules described herein (suchas any of systems 300, 400, 500 a, or 500 b, or any other systemdescribed herein), or any element thereof, may be environmentallycontrolled. For instance, the systems may be maintained at a specifiedtemperature or humidity. The systems (or any element thereof) may bemaintained at a temperature of at least 20 degrees Celsius (° C.), atleast 25° C., at least 30° C., at least 35° C., at least 40° C., atleast 45° C., at least 50° C., at least 55° C., at least 60° C., atleast 65° C., at least 70° C., at least 75° C., at least 80° C., atleast 85° C., at least 90° C., at least 95° C., at least 100° C., atmost 100° C., at most 95° C., at most 90° C., at most 85° C., at most80° C., at most 75° C., at most 70° C., at most 65° C., at most 60° C.,at most 55° C., at most 50° C., at most 45° C., at most 40° C., at most35° C., at most 30° C., at most 25° C., at most 20° C., or at atemperature that is within a range defined by any two of the precedingvalues. Different elements of the system may be maintained at differenttemperatures or within different temperature ranges, such as thetemperatures or temperature ranges described herein. Elements of thesystem may be set at temperatures above the dewpoint to preventcondensation. Elements of the system may be set at temperatures belowthe dewpoint to collect condensation.

The systems (or any element thereof) may be maintained at a relativehumidity of at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at most 100%, at most 95%, at most 90%, at most 85%, at most80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, atmost 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most25%, at most 20%, at most 15%, at most 10%, at most 5%, or a relativehumidity that is within a range defined by any two of the precedingvalues. The systems (or any element thereof) may be contained within asealed container, housing, or chamber that insulates the system (or anyelement thereof) from the external environment, allowing for the controlof the temperature or humidity. An environmental unit (e.g.,humidifiers, heaters, heat exchangers, compressors, etc.) may beconfigured to regulate one or more operating conditions in eachenvironment. In some instances, each environment may be regulated byindependent environmental units. In some instances, a singleenvironmental unit may regulate a plurality of environments. In someinstances, a plurality of environmental units may, individually orcollectively, regulate the different environments. An environmental unitmay use active methods or passive methods to regulate the operatingconditions. For example, the temperature may be controlled using heatingor cooling elements. The humidity may be controlled using humidifiers ordehumidifiers. In some instances, a part of the internal environmentwithin the container or chamber may be further controlled from otherparts of the internal environment. Different parts may have differentlocal temperatures, pressures, and/or humidity. For example, theinternal environment may comprise a first internal environment and asecond internal environment separated by a seal. In some instances, theseal may comprise an immersion objective lens. For example, an immersionobjective lens may be part of a seal that separates the internalenvironment in the container into a first internal environment having100% (or substantially 100%) humidity and a second environment havingone or more of an ambient temperature, pressure or humidity. Theimmersion objective lens may be in contact with one or more of adetector and imaging lens.

Optical Systems for Imaging a Rotating Substrate

For a substrate exhibiting a smooth, stable rotational motion, it may besimpler or more cost-effective to image the substrate using a rotationalmotion system instead of a rectilinear motion system. Rotational motion,as used herein, may generally refer to motion in a polar coordinatesystem that is predominantly in an angular direction. Prior opticalimaging systems have utilized time delay and integration (TDI) camerasto achieve high duty cycles and maximum integration times per fieldpoint. A TDI camera may use a detection principle similar to a chargecoupled device (CCD) camera. Compared to a CCD camera, the TDI cameramay shift electric charge, row by row, across a sensor at the same rateas an image traverses the focal plane of the camera. In this manner, theTDI camera may allow longer image integration times while reducingartifacts such as blurring that may be otherwise associated with longimage exposure times. A TDI camera may perform integration whilesimultaneously reading out and may therefore have a higher duty cyclethan a camera that performs these functions in a serial manner. Use of aTDI camera to extend integration times may be important for highthroughput fluorescent samples, which may be limited in signalproduction by fluorescent lifetimes. For instance, alternative imagingtechniques, such as point scanning, may be precluded from use in highthroughput systems as it may not be possible to acquire an adequatenumber of photons from a point in the limited amount of integration timerequired for high speeds due to limits imposed by fluorescence lifetimesof dye molecules.

Prior TDI detection schemes may be limited in their applicability to theimaging of rotating systems, such as the rotating nucleic acidsequencing systems described herein. When scanning a curved path, suchas the curved path generated by the rotating systems described herein, aTDI sensor may only be able to shift charge (commonly referred to asclocking or line triggering) at the correct rate for a single velocity.For instance, the TDI sensor may only be able to clock at the correctrate along an arc located at a particular distance from the center ofrotation. Locations at smaller distances from the center of rotation mayclock too quickly, while locations at smaller distances from the centerof rotation may clock too slowly. In either case, the mismatch betweenthe rotational speed of the rotating system and the clock rate of theTDI sensor may cause blurring that varies with the distance of alocation from the center of the rotating system. This effect may bereferred to as tangential velocity blur. The tangential velocity blurmay produce an image distortion of a magnitude a defined by equation(2):

$\begin{matrix}{\sigma = {\frac{hw}{2R} = \frac{A}{2R}}} & (2)\end{matrix}$

Here, h, w, and A are the effective height, width, and area,respectively, of the TDI sensor projected to the object plan. R is thedistance of the center of the field from the center of the rotatingsystem. The effective height, width, and area of the sensor are theheight, width, and area, respectively, that produce signal. In the caseof fluorescence imaging, the effective height, width, and area of thesensor may be the height, width, and area, respectively, that correspondto illuminated areas on the sample. In addition to the tangentialvelocity blur effect, Equation (2) implies that increasing sensor area,which may be a goal of many imaging systems, may introduce imagingcomplications for TDI imaging of rotating systems. Consequently, priorTDI systems may require small image sensors to image rotating systemsand may thus be unfit for simultaneous high-sensitivity andhigh-throughput imaging of such systems.

Described herein are systems and methods for imaging rotating systemsthat can address at least the abovementioned problems. The systems andmethods described herein may benefit from higher efficiency, such asfrom faster imaging time.

FIG. 7 shows an optical system 700 for continuous area scanning of asubstrate during rotational motion of the substrate. The term“continuous area scanning (CAS),” as used herein, generally refers to amethod in which an object in relative motion is imaged by repeatedly,electronically or computationally, advancing (clocking or triggering) anarray sensor at a velocity that compensates for object motion in thedetection plane (focal plane). CAS can produce images having a scandimension larger than the field of the optical system. TDI scanning maybe an example of CAS in which the clocking entails shiftingphotoelectric charge on an area sensor during signal integration. For aTDI sensor, at each clocking step, charge may be shifted by one row,with the last row being read out and digitized. Other modalities mayaccomplish similar function by high speed area imaging and co-additionof digital data to synthesize a continuous or stepwise continuous scan.

The optical system may comprise one or more sensors 710. As shown, inFIG. 7, the sensors may be optically projected to the sample. Theoptical system may comprise one or more optical elements, such as theoptical element 810 described in the context of FIG. 8. The system maycomprise a plurality of sensors, such as at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, or at least 1,000 sensors. The system may comprise a at least 2, atleast 4, at least 8, at least 16, at least 32, at least 64, at least128, at least 256, at least 512, or at least 1,024 sensors. Theplurality of sensors may be the same type of sensor or different typesof sensors. Alternatively, the system may comprise at most about 1000,500, 200, 100, 50, 20, 10, 5, 2, or fewer sensors. Alternatively, thesystem may comprise at most about 1024, 512, 256, 128, 64, 32, 16, 8, 4,2, or fewer sensors. The system may comprise a number of sensors that iswithin a range defined by any two of the preceding values. The sensorsmay comprise image sensors. The sensors may comprise CCD cameras. Thesensors may comprise CMOS cameras. The sensors may comprise TDI cameras.The sensors may comprise pseudo-TDI rapid frame rate sensors. Thesensors may comprise CMOS TDI or hybrid cameras. The sensors may beintegrated together in a single package. The sensors may be integratedtogether in a single semiconductor substrate. The system may furthercomprise any optical source described herein (not show in FIG. 7).

The sensors may be configured to detect an image from a substrate, suchas the substrate 310 described herein, during rotational motion of thesubstrate. The rotational motion may be with respect to an axis of thesubstrate. The axis may be an axis through the center of the substrate.The axis may be an off-center axis. The substrate may be configured torotate at any rotational speed described herein. The rotational motionmay comprise compound motion. The compound motion may comprise rotationand an additional component of radial motion. The compound motion may bea spiral (or substantially spiral). The compound motion may be a ring(or substantially ring-like).

Each sensor may be located at a focal plane in optical communicationwith the substrate. The focal plane may be the approximate plane in animaging system (e.g., CAS sensor) at which an image of a region of thesubstrate forms. The focal plane may be segmented into a plurality ofregions, such as at least 2, at least 5, at least 10, at least 20, atleast 50, at least 100, at least 200, at least 500, or at least 1000regions. The focal plane may be segmented into at least 2, at least 4,at least 8, at least 16, at least 32, at least 64, at least 128, atleast 256, at least 512, or at least 1,024 regions. The focal plane maybe segmented into a number of regions that is within a range defined byany two of the preceding values. The focal plane may be segmented into aplurality of regions along an axis substantially normal to a projecteddirection of the rotational motion. An angle between the axis and theprojected direction of the rotational motion may be no more than 1degree, no more than 2 degrees, no more than 3 degrees, no more than 4degrees, no more than 5 degrees, no more than 6 degrees, no more than 7degrees, no more than 8 degrees, no more than 9 degrees, no more than 10degrees, no more than 11 degrees, no more than 12 degrees, no more than13 degrees, no more than 14 degrees, or no more than 15 degrees fromnormal, or an angle that is within a range defined by any two of thepreceding values. The focal plane may be segmented into a plurality ofregions along an axis parallel to a projected direction of therotational motion. The focal plane may be spatially segmented. Forinstance, the focal plane may be segmented by abutting or otherwisearranging a plurality of sensors in a single focal plane and clockingeach of the sensors independently.

Alternatively or in combination, the focal plane may be segmented byoptically splitting the focal plane into a plurality of separate paths,each of which may form a sub-image on an independent sensor of theplurality of sensors and which may be clocked independently. The focalpath may be optically split using one or more optical elements, such asa lens array, mirror, or prism. Each sensor of the plurality of sensorsmay be in optical communication with a different region of the rotatingsubstrate. For instance, each sensor may image a different region of therotating substrate. Each sensor of the plurality of sensors may beclocked at a rate appropriate for the region of the rotating substrateimaged by the sensor, which may be based on the distance of the regionfrom the center of the rotating substrate or the tangential velocity ofthe region.

One or more of the sensors may be configured to be in opticalcommunication with at least 2 of the plurality of regions in the focalplane. One or more of the sensors may comprise a plurality of segments.Each segment of the plurality of segments may be in opticalcommunication with a region of the plurality of regions. Each segment ofthe plurality of segments may be independently clocked. The independentclocking of a segment may be linked to a velocity of an image in anassociated region of the focal plane. The independent clocking maycomprise TDI line rate or pseudo-TDI frame rate.

The system may further comprise a controller (not shown). The controllermay be operatively coupled to the one or more sensors. The controllermay be programmed to process optical signals from each region of therotating substrate. For instance, the controller may be programmed toprocess optical signals from each region with independent clockingduring the rotational motion. The independent clocking may be based atleast in part on a distance of each region from a projection of the axisand/or a tangential velocity of the rotational motion. The independentclocking may be based at least in part on the angular velocity of therotational motion. While a single controller has been described, aplurality of controllers may be configured to, individually orcollectively, perform the operations described herein.

FIG. 8A shows an optical system 800 for imaging a substrate duringrotational motion of the substrate using tailored optical distortions.The optical system may comprise one or more sensors 710. The one or moresensors may comprise any sensors described herein. The optical systemmay comprise any optical sources described herein (not shown in FIG.8A).

The sensors may be configured to detect an image from a substrate, suchas the substrate 310 described herein, during rotational motion of thesubstrate. The rotational motion may be with respect to an axis of thesubstrate. The axis may be an axis through the center of the substrate.The axis may be an off-center axis. The substrate may be configured torotate at any rotational speed described herein.

The system 800 may further comprise an optical element 810. The opticalelement may be in optical communication with the sensor. The opticalelement may be configured to direct optical signals from the substrateto the sensor. The optical element may produce an optical magnificationgradient across the sensor. At least one of the optical element and thesensor may be adjustable. For instance, at least one of the opticalelement and the sensor may be adjustable to generate an opticalmagnification gradient across the sensor. The optical magnificationgradient may be along a direction substantially perpendicular to aprojected direction of the rotational motion of the substrate. Theoptical element may be configured to rotate, tilt, or otherwise bepositioned to engineer the optical magnification gradient. The opticalelement may produce a magnification that scales approximately as theinverse of the distance to the axis of the substrate. The magnificationgradient may be produced by selecting a relative orientation of thesubstrate, optical element, and sensor. For instance, the magnificationgradient may be produced by tilting the object and image planes as shownin FIG. 8A. The magnification gradient may display geometric properties.For instance, a ratio of a first optical magnification of a first regionat a minimum distance from the center of the substrate to a secondoptical magnification of a second region at a maximum distance from thecenter of the substrate may be substantially equal to a ratio of themaximum distance to the minimum distance. In this manner, the first andsecond optical magnifications may be in the same ratio as the radii oftheir respective sample regions. Although the system 800 as shownincludes a single optical element 810, the system 800 may include aplurality of optical elements, such as at least 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 100, or more optical elements. Various arrangementsor configurations of optical elements may be employed. For example, thesystem 800 may include a lens and a mirror for directing light.

The optical element may be a lens. The lens may be a field lens. Thelens may be a cylindrical lens (for instance, as shown in FIG. 8B). Thecylindrical lens may be plano-cylindrical. The lens may be plano-concaveor plano-convex. The cylindrical lens may have a positive or negativecurvature. The curvature of the cylindrical lens may vary. The curvatureof the cylindrical lens may vary in a direction perpendicular to aprojected direction of rotational motion. The shape of a surface of thelens may be conical. The lens may be tilted with respect to the sensor,thereby producing an anamorphic magnification gradient. The tilt of thelens may be adjustable, thereby producing an adjustable anamorphicmagnification gradient.

FIG. 8B shows an example of induced tailored optical distortions using acylindrical lens. As shown in FIG. 8B, a cylindrical lens may have afirst side A and a second side B. The first side A may be located closerto an image sensor (such as a TDI camera sensor described herein) thanthe second side B. Such a configuration may be achieved by tilting thecylindrical lens in relation to the image sensor. In this manner, thecylindrical lens may direct light to different locations on the imagesensor, with light passing through side B being directed moredivergently than light passing through side A. In this manner, thecylindrical lens may provide an anamorphic magnification gradient acrossthe image sensor, as depicted in FIG. 8B.

Tilting of the lens may provide an anamorphic magnification gradientacross the sensor. The tilt and hence anamorphic gradient may be in adirection substantially perpendicular to the image motion on the sensor.The tilt of the lens may be adjustable. The adjustment may be automaticby using a controller. The adjustment may be coupled to the radius ofthe scanned substrate region relative to the substrate axis of rotation.The ratio of the minimum to maximum anamorphic magnification may beexactly or approximately in the ratio of the minimum to maximumprojected radii relative to the substrate axis of rotation.

Alternatively or in combination, a gradient in the radius of curvatureof the lens may provide an anamorphic magnification gradient across thesensor. The curvature gradient may be in a direction substantiallydirection perpendicular to the image motion on the sensor.

The system may further comprise a controller (not shown). The controllermay be operatively coupled to the sensor and the optical element. Thecontroller may be programmed to direct the adjustment of at least one ofthe sensor and the optical element to generate an optical magnificationgradient across the sensor. The magnification gradient may be generatedalong a direction substantially perpendicular to a projected directionof the rotational motion. The controller may be programmed to directadjustment of the sensor and/or the optical element to produce ananamorphic optical magnification gradient. The optical magnificationgradient may be across the sensor in a direction substantiallyperpendicular to a projected direction of the rotational motion. Thecontroller may be programmed to direct rotation or tilt of the opticalelement. The controller may be programmed to direct adjustment of themagnification gradient. For instance, the controller may be programmedto direct adjustment of the magnification gradient at least in part on aradial range of a field dimension relative to a projection about theaxis of the substrate. The controller may be programmed to subject therotational motion to the substrate. While a single controller has beendescribed, a plurality of controllers may be configured to, individuallyor collectively, perform the operations described herein.

The optical systems described herein may utilize multiple scan heads.The multiple scan heads may be operated in parallel along differentimaging paths. For instance, the scan heads may be operated to produceinterleaved spiral scans, nested spiral scans, interleaved ring scans,nested ring scans, or a combination thereof.

FIG. 9A shows a first example of an interleaved spiral imaging scan. Afirst region of a scan head may be operated along a first spiral path910 a. A second region of a scan head may be operated along a secondspiral path 920 a. A third region of a scan head may be operated along athird spiral path 930 a. Each of the first, second, and third regionsmay be independently clocked. The scan head may comprise any opticalsystems described herein. The use of multiple imaging scan paths mayincrease imaging throughput by increasing imaging rate.

FIG. 9B shows a second example of an interleaved spiral imaging scan. Afirst scan head may be operated along a first spiral path 910 b. Asecond scan head may be operated along a second spiral path 920 b. Athird scan head may be operated along a third spiral path 930 b. Each ofthe first, second, and third scan heads may be independently clocked orclocked in unison. Each of the first, second, and third scan heads maycomprise any optical systems described herein. The use of multipleimaging scan paths may increase imaging throughput by increasing netimaging rate. Throughput of the optical system can be multiplied byoperating many scan heads of a field width in parallel. For example,each scan head may be fixed at a different angle relative to the centerof substrate rotation.

FIG. 9C shows an example of a nested spiral imaging scan. A first scanhead may be operated along a first spiral path 910 c. A second scan headmay be operated along a second spiral path 920 c. A third scan head maybe operated along a third spiral path 930 c. Each of the first, second,and third scan heads may be independently clocked. Each of the first,second, and third scan heads may comprise any optical systems describedherein. The use of multiple imaging scan paths may increase imagingthroughput by increasing imaging rate. The scan heads may move togetherin the radial direction. Throughput of the optical system can bemultiplied by operating many scan heads of a field width in parallel.For example, each scan head may be fixed at a different angle. The scansmay be in discrete rings rather or spirals.

While FIGS. 9A-9C illustrate three imaging paths, there may be anynumber of imaging paths and any number of scan heads. For example, theremay be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more imaging pathsor scan heads. Alternatively, there may be at most about 10, 9, 8, 7, 6,5, 4, 3, 2, or less imaging paths or scan heads. Each scan head may beconfigured to receive light having a wavelength within a givenwavelength range. For instance, the first scan head may be configured toreceive first light having a wavelength within a first wavelength range.The second scan head may be configured to receive second light having awavelength within a second wavelength range. The third scan head may beconfigured to receive third light having a wavelength within a thirdwavelength range. Similarly, fourth, fifth, sixth, seventh, eighth,ninth, or tenth scan heads may be configured to receive fourth, fifth,sixth, seventh, eighth, ninth, or tenth light, respectively, each of thefourth, fifth, sixth, seventh, eighth, ninth, or tenth light having awavelength within a fourth, fifth, sixth, seventh, eighth, ninth, ortenth wavelength range, respectively. The first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may beidentical. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may partially overlap. Any 2,3, 4, 5, 6, 7, 8, 9, or 10 of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may bedistinct. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may be in the ultraviolet,visible, or near infrared regions of the electromagnetic spectrum. Eachof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth wavelength ranges may comprise a wavelength emitted by afluorophore, dye, or quantum dot described herein. In this manner, thesystem may be configured to detect optical signals from a plurality offluorophores, dyes, or quantum dots.

FIG. 10 shows a nested circular imaging scan. A first scan head 1005 maybe operated along a first approximately circular path 1010. A secondscan head 1015 may be operated along a second approximately circularpath 1020. A third scan head 1025 may be operated along a thirdapproximately circular path 1030. A fourth scan head 1035 may beoperated along a fourth approximately circular path 1040. A fifth scanhead 1045 may be operated along a fifth approximately circular path1050. A sixth scan head 1055 may be operated along a sixth approximatelycircular path 1060. Each of the first, second, third, fourth, fifth, andsixth scan heads may be independently clocked. Each of the first,second, third, fourth, fifth, and sixth scan heads may comprise anyoptical systems described herein. Each of the first, second, third,fourth, fifth, and sixth scan heads may be configured to remain in afixed location during scanning of a substrate. Alternatively, one ormore of the first, second, third, fourth, fifth, and sixth scan headsmay be configured to move during scanning of a substrate. The use of aplurality of scan heads imaging along approximately circular imagingpaths may greatly increase imaging throughput. For instance, theconfiguration of scan heads depicted in FIG. 10 may allow alladdressable locations on a substrate to be imaged during a singlerotation of the substrate. Such a configuration may have the additionaladvantage of simplifying the mechanical complexity of an imaging systemby requiring only one scanning motion (e.g., the rotation of thesubstrate).

While FIG. 10 illustrates six imaging paths and six scan heads, theremay be any number of imaging paths and any number of scan heads. Forexample, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreimaging paths or scan heads. Alternatively, there may be at most about10, 9, 8, 7, 6, 5, 4, 3, 2, or less imaging paths or scan heads. Eachscan head may be configured to receive light having a wavelength withina given wavelength range. For instance, the first scan head may beconfigured to receive first light having a wavelength within a firstwavelength range. The second scan head may be configured to receivesecond light having a wavelength within a second wavelength range. Thethird scan head may be configured to receive third light having awavelength within a third wavelength range. The fourth scan head may beconfigured to receive fourth light having a wavelength within a fourthwavelength range. The fifth scan head may be configured to receive fifthlight having a wavelength within a fifth wavelength range. The sixthscan head may be configured to receive sixth light having a wavelengthwithin a sixth wavelength range. Similarly, seventh, eighth, ninth, ortenth scan heads may be configured to receive seventh, eighth, ninth, ortenth light, respectively, each of the seventh, eighth, ninth, or tenthlight having a wavelength within a seventh, eighth, ninth, or tenthwavelength range, respectively. The first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may beidentical. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may partially overlap. Any 2,3, 4, 5, 6, 7, 8, 9, or 10 of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may bedistinct. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may be in the ultraviolet,visible, or near infrared regions of the electromagnetic spectrum. Eachof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth wavelength ranges may comprise a wavelength emitted by afluorophore, dye, or quantum dot described herein. In this manner, thesystem may be configured to detect optical signals from a plurality offluorophores, dyes, or quantum dots.

FIG. 11 shows a cross-sectional view of an immersion optical system1100. The system 1100 may be used to optically image the substratesdescribed herein. The system 1100 may be integrated with any otheroptical system or system for nucleic acid sequencing described herein(such as any of systems 300, 400, 500 a, 500 b, 700, or 800), or anyelement thereof. The system may comprise an optical imaging objective1110. The optical imaging objective may be an immersion optical imagingobjective. The optical imaging objective may be configured to be inoptical communication with a substrate, such as substrate 310 describedherein. The optical imaging objective may be configured to be in opticalcommunication with any other optical elements described herein. Theoptical imaging objective may be partially or completely surrounded byan enclosure 1120. The enclosure may partially or completely surround asample-facing end of the optical imaging objective. The enclosure andfluid may comprise an interface between the atmosphere in contact withthe substrate and the ambient atmosphere. The atmosphere in contact withthe substrate and the ambient atmosphere may differ in relativehumidity, temperature, and/or pressure. The enclosure may have agenerally cup-like shape or form. The enclosure may be any container.The enclosure may be configured to contain a fluid 1140 (such as wateror an aqueous or organic solution) in which the optical imagingobjective is to be immersed. The enclosure may be configured to maintaina minimal distance 1150 between the substrate and the enclosure in orderto avoid contact between the enclosure and the substrate during rotationof the substrate. The minimal distance may be at least 100 nm, at least200 nm, at least 500 nm, at least 1 μm, at least 2 μm, at least 5 μm, atleast 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least200 μm, at least 500 μm, at least 1 mm, or a distance that is within arange defined by any two of the preceding values. Even with a minimaldistance, the enclosure may contain the fluid due to surface tensioneffects. The system may comprise a fluid flow tube 1130 configured todeliver fluid to the inside of the enclosure. The fluid flow tube may beconnected to the enclosure through an adaptor 1135. The adaptor maycomprise a threaded adaptor, a compression adaptor, or any otheradaptor. An electrical field application unit (not shown) can beconfigured to regulate a hydrophobicity of one or more surfaces of acontainer to retain at least a portion of the fluid contacting theimmersion objective lens and the open substrate, such as by applying anelectrical field.

The fluid may be in contact with the substrate. The optical imagingobjective and enclosure may be configured to provide a physical barrierbetween a first location in which chemical processing operations areperformed and a second location in which detection operations areperformed. In this manner, the chemical processing operations and thedetection operations may be performed with independent operationconditions and contamination of the detector may be avoided. The firstand second locations may have different humidities, temperatures,pressures, or atmospheric admixtures.

A system of the present disclosure may be contained in a container orother closed environment. For example, a container may isolate aninternal environment 1160 from an external environment 1170. Theinternal environment 1160 may be controlled such as to localizetemperature, pressure, and/or humidity, as described elsewhere herein.In some instances, the external environment 1170 may be controlled. Insome instances, the internal environment 1160 may be furtherpartitioned, such as via, or with aid of, the enclosure 1120 toseparately control parts of the internal environment (e.g., firstinternal environment for chemical processing operations, second internalenvironment for detection operations, etc.). The different parts of theinternal environment may be isolated via a seal. For example, the sealmay comprise the immersion objective described herein.

System Architectures for High-Throughput Processing

The nucleic acid sequencing systems and optical systems described herein(or any elements thereof) may be combined in a variety of architectures.

FIG. 12A shows an architecture for a system 1200 a comprising astationary substrate and moving fluidics and optics. The system 1200 amay comprise substrate 310 described herein. The substrate may beconfigured to rotate, as described herein. The substrate may be adheredor otherwise affixed to a chuck (not shown in FIG. 12A), as describedherein. The system may further comprise fluid channel 330 and fluidoutlet port 335 described herein, and/or any other fluid channel andfluid outlet port described herein. The fluid channel and fluid outletport may be configured to dispense any solution described herein. Thefluid channel and fluid outlet port may be configured to move 1215 arelative to the substrate. For instance, the fluid channel and fluidoutlet port may be configured to move to a position above (such as nearthe center of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The fluidchannel and fluid outlet port may be configured to move to a positionaway from the substrate during the period in which the fluid channel andfluid outlet port are not dispensing a solution. Alternatively, thereverse may apply. The system may further comprise optical imagingobjective 1110 described herein. The optical imaging objective may beconfigured to move 1210 a relative for the substrate. For instance, theoptical imaging objective may be configured to move to a position above(such as near the center of) the substrate during periods of time inwhich the substrate is being imaged. The optical imaging objective maybe configured to move to a position away from the substrate during theperiod in which the substrate is not being imaged. The system mayalternate between dispensing of solutions and imaging, allowing rapidsequencing of the nucleic acids attached to the substrate using thesystems and methods described herein.

FIG. 12B shows an architecture for a system 1200 b comprising a movingsubstrate and stationary fluidics and optics. The system 1200 b maycomprise substrate 310 described herein. The substrate may be configuredto rotate, as described herein. The substrate may be adhered orotherwise affixed to a chuck (not shown in FIG. 12B), as describedherein. The system may further comprise fluid channel 330 and fluidoutlet port 335 described herein, or any other fluid channel and fluidoutlet port described herein. The fluid channel and fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise optical imaging objective 1110 described herein.The fluid channel, fluid outlet port, and optical imaging objective maybe stationary. The substrate may be configured to move 1210 b relativeto the fluid channel, fluid outlet port, and optical imaging objective.For instance, the substrate may be configured to move to a position suchthat the fluid channel and fluid outlet port are above (such as near thecenter of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The substratemay be configured to move to a position away from the fluid channel andfluid outlet port during the period in which the fluid channel and fluidoutlet port are not dispensing a solution. The substrate may beconfigured to radially scan the objective over the substrate duringperiods of time in which the substrate is being imaged. The substratemay be configured to move to a position away from the optical imagingobjective during the period in which the substrate is not being imaged.The system may alternate between dispensing of solutions and imaging,allowing rapid sequencing of the nucleic acids attached to the substrateusing the systems and methods described herein.

FIG. 12C shows an architecture for a system 1200 c comprising aplurality of stationary substrates and moving fluidics and optics. Thesystem 1200 c may comprise first and second substrates 310 a and 310 b.The first and second substrates may be similar to substrate 310described herein. The first and second substrates may be configured torotate, as described herein. The first and second substrates may beadhered or otherwise affixed to first and second chucks (not shown inFIG. 12C), as described herein. The system may further comprise firstfluid channel 330 a and first fluid outlet port 335 a. First fluidchannel 330 a may be similar to fluid channel 330 described herein orany other fluid channel described herein. First fluid outlet port 335 amay be similar to fluid outlet port 335 described herein or any otherfluid outlet port described herein. The system may further comprisesecond fluid channel 330 b and second fluid outlet port 335 b. Secondfluid channel 330 b may be similar to fluid channel 330 described hereinor any other fluid channel described herein. Second fluid outlet port335 a may be similar to fluid outlet port 335 described herein or anyother fluid outlet port described herein. The first fluid channel andfirst fluid outlet port may be configured to dispense any solutiondescribed herein. The second fluid channel and second fluid outlet portmay be configured to dispense any solution described herein.

The system may further comprise optical imaging objective 1110. Opticalimaging objective 1110 may be configured to move 1210 c relative to thefirst and second substrates. For instance, the optical imaging objectivemay be configured to move to a position above (such as near the centerof, or radially scanning) the first substrate during periods of time inwhich the first fluid channel and first fluid outlet port are notdispensing a solution to the second substrate (and during which thefirst substrate is to be imaged). The optical imaging objective may beconfigured to move to a position away from the first substrate duringthe period in which the first fluid channel and first fluid outlet portare dispensing a solution. The optical imaging objective may beconfigured to move to a position above (such as near the center of, orradially scanning) the second substrate during periods of time in whichthe second fluid channel and second fluid outlet port are not dispensinga solution to the second substrate (and during which the secondsubstrate is to be imaged). The optical imaging objective may beconfigured to move to a position away from the second substrate duringthe period in which the second fluid channel and second fluid outletport are dispensing a solution.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the optical imaging objective maybe moved from the second substrate to the first substrate. A solutionmay then be dispensed to the second substrate during a period of time inwhich the first substrate is being imaged. This alternating pattern ofdispensing and imaging may be repeated, allowing rapid sequencing of thenucleic acids attached to the first and second substrates using thesystems and methods described herein. The alternating pattern ofdispensing and imaging may speed up the sequencing by increasing theduty cycle of the imaging process or the solution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 12C,system 1200 c may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and/or at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. Each optical imagingobjective may be moved between substrates as described herein.

FIG. 12D shows an architecture for a system 1200 d comprising aplurality of moving substrates on a rotary stage and stationary fluidicsand optics. The system 1200 d may comprise first and second substrates310 a and 310 b. The first and second substrates may be similar tosubstrate 310 described herein. The first and second substrates may beconfigured to rotate, as described herein. The first and secondsubstrates may be adhered or otherwise affixed to first and secondchucks (not shown in FIG. 12D), as described herein. The first andsecond substrates may be affixed to a rotating stage 1220 d (such asapproximately at opposing ends of the rotating stage). The rotatingstage may be configured to rotate about an axis. The axis may be an axisthrough the center of the substrate. The axis may be an off-center axis.The rotating stage may approximately scan the radius of the substrate310 b. The system may further comprise fluid channel 330 and fluidoutlet port 335. The fluid channel and fluid outlet port may beconfigured to dispense any solution described herein. The system mayfurther comprise optical imaging objective 1110. A longitudinal axis ofthe imaging objective 1110 may not be coincident with a central axis ofthe second substrate 310 b (although this is difficult to distinguish inFIG. 12D). The imaging objective 1110 may be positioned at some distancefrom a center of the second substrate 310 b.

The rotating stage may be configured to alter the relative positions ofthe first and second substrates to carry out different sequencingoperations. For instance, the rotating stage may be configured to rotatesuch that the optical imaging objective is in a position above (such asnear the center of, or radially scanning) the first substrate duringperiods of time in which the fluid channel and fluid outlet port are notdispensing a solution to the first substrate (and during which the firstsubstrate is to be imaged). The rotating stage may be configured torotate such that the optical imaging objective is away from the firstsubstrate during the period in which the fluid channel and fluid outletport are dispensing a solution to the first substrate. The rotatingstage may be configured to rotate such that the optical imagingobjective is in a position above (such as near the center of, orradially scanning) the second substrate during periods of time in whichthe fluid channel and fluid outlet port are not dispensing a solution tothe second substrate (and during which the second substrate is to beimaged). The rotating stage may be configured to rotate such that theoptical imaging objective is away from the second substrate during theperiod in which the fluid channel and fluid outlet port are dispensing asolution to the second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the rotating stage may be rotatedsuch that a solution may be dispensed to the second substrate during aperiod of time in which the first substrate is being imaged. Thisalternating pattern of dispensing and imaging may be repeated, allowingrapid sequencing of the nucleic acids attached to the first and secondsubstrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, one fluid channel, onefluid outlet port, and one optical imaging objective in FIG. 12D, system1200 d may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The rotating stagemay be rotated to place any substrate under any fluid channel, fluidoutlet port, or optical imaging objective at any time.

FIG. 12E shows an architecture for a system 1200 e comprising aplurality of stationary substrates and moving optics. The system 1200 dmay comprise first and second substrates 310 a and 310 b. The first andsecond substrates may be similar to substrate 310 described herein. Thefirst and second substrates may be configured to rotate, as describedherein. The first and second substrates may be adhered or otherwiseaffixed to first and second chucks (not shown in FIG. 12E), as describedherein. The system may further comprise first fluid channel 330 a andfirst fluid outlet port 335 a. First fluid channel 330 a may be similarto fluid channel 330 described herein or any other fluid channeldescribed herein. First fluid outlet port 335 a may be similar to fluidoutlet port 335 described herein or any other fluid outlet portdescribed herein. The first fluid channel and first fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise second fluid channel 330 b and second fluid outletport 335 b. Second fluid channel 330 b may be similar to fluid channel330 described herein or any other fluid channel described herein. Secondfluid outlet port 335 b may be similar to fluid outlet port 335described herein or any other fluid outlet port described herein. Thesecond fluid channel and second fluid outlet port may be configured todispense any solution described herein.

The system may further comprise optical imaging objective 1110. Theoptical imaging objective may be attached to an imaging arm 1230 e. Theoptical imaging objective may be configured to move 1220 e along theoptical imaging arm to image an entire area of the first or secondsubstrate. The optical imaging arm may be configured to rotate 1210 e.The optical imaging arm may be configured to rotate such that theoptical imaging objective is in a position above (such as near thecenter of, or radially scanning) the first substrate during periods oftime in which the first fluid channel and first fluid outlet port arenot dispensing a solution to the first substrate (and during which thefirst substrate is to be imaged). The optical imaging arm may beconfigured to rotate such that the optical imaging objective is awayfrom the first substrate during the period in which the first fluidchannel and first fluid outlet port are dispensing a solution to thefirst substrate. The optical imaging arm may be configured to rotatesuch that the optical imaging objective is in a position above (such asnear the center of, or radially scanning) the second substrate duringperiods of time in which the second fluid channel and second fluidoutlet port are not dispensing a solution to the second substrate (andduring which the second substrate is to be imaged). The optical imagingarm may be configured to rotate such that the optical imaging objectiveis away from the second substrate during the period in which the secondfluid channel and second fluid outlet port are dispensing a solution tothe second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the optical imaging arm may berotated such that a solution may be dispensed to the second substrateduring a period of time in which the first substrate is being imaged.This alternating pattern of dispensing and imaging may be repeated,allowing rapid sequencing of the nucleic acids attached to the first andsecond substrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 12E,system 1200 e may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The optical imagingarm may be rotated to place any substrate under any fluid channel, fluidoutlet port, or optical imaging objective at any time.

FIG. 12F shows an architecture for a system 1200 f comprising aplurality of moving substrates and stationary fluidics and optics. Thesystem 1200 f may comprise first and second substrates 310 a and 310 b.The first and second substrates may be similar to substrate 310described herein. The first and second substrates may be configured torotate, as described herein. The first and second substrates may beadhered or otherwise affixed to first and second chucks (not shown inFIG. 12F), as described herein. The first and second substrates may beaffixed to opposing ends of a moving stage 1220 f. The moving stage maybe configured to move 1210 f. The system may further comprise firstfluid channel 330 a and first fluid outlet port 335 a. First fluidchannel 330 a may be similar to fluid channel 330 described herein orany other fluid channel described herein. First fluid outlet port 335 amay be similar to fluid outlet port 335 described herein or any otherfluid outlet port described herein. The first fluid channel and firstfluid outlet port may be configured to dispense any solution describedherein. The system may further comprise second fluid channel 330 b andsecond fluid outlet port 335 b. Second fluid channel 330 b may besimilar to fluid channel 330 described herein or any other fluid channeldescribed herein. Second fluid outlet port 335 b may be similar to fluidoutlet port 335 described herein or any other fluid outlet portdescribed herein. The second fluid channel and second fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise optical imaging objective 1110.

The moving stage may be configured to move such that the optical imagingobjective is in a position above (such as near the center of, orradially scanning) the first substrate during periods of time in whichthe first fluid channel and first fluid outlet port are not dispensing asolution to the first substrate (and during which the first substrate isto be imaged). The moving stage may be configured to move such that theoptical imaging objective is away from the first substrate during theperiod in which the first fluid channel and first fluid outlet port aredispensing a solution to the first substrate. The moving stage may beconfigured to move such that the optical imaging objective is in aposition above (such as near the center of, or radially scanning) thesecond substrate during periods of time in which the second fluidchannel and second fluid outlet port are not dispensing a solution tothe second substrate (and during which the second substrate is to beimaged). The moving stage may be configured to move such that theoptical imaging objective is away from the second substrate during theperiod in which the second fluid channel and second fluid outlet portare dispensing a solution to the second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the moving stage may move suchthat a solution may be dispensed to the second substrate during a periodof time in which the first substrate is being imaged. This alternatingpattern of dispensing and imaging may be repeated, allowing rapidsequencing of the nucleic acids attached to the first and secondsubstrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 12F,system 1200 f may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The moving stage maymove so as to place any substrate under any fluid channel, fluid outletport, or optical imaging objective at any time.

FIG. 12G shows an architecture for a system 1200 g comprising aplurality of substrates moved between a plurality of processing bays.The system 1200 g may comprise first, second, third, and fourthsubstrates 310 a, 310 b, 310 c, 310 d, and 310 e, respectively. Thefirst, second, third, fourth, and fifth substrates may be similar tosubstrate 310 described herein. The first, second, third, fourth, andfifth substrates may be configured to rotate, as described herein. Thefirst, second, third, fourth, and fifth substrates may be adhered orotherwise affixed to first, second, third, fourth, and fifth chucks (notshown in FIG. 12G), respectively, as described herein.

The system may further comprise first fluid channel 330 a and firstfluid outlet port 335 a. First fluid channel 330 a may be similar tofluid channel 330 described herein or any other fluid channel describedherein. First fluid outlet port 335 a may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The first fluid channel and first fluid outlet port may beconfigured to dispense any solution described herein. The first fluidchannel and first fluid outlet port may be regarded as a firstprocessing bay. The first processing bay may be configured to perform afirst processing operation, such as dispensing of a first solution toany of the first, second, third, fourth, or fifth substrates.

The system may further comprise second fluid channel 330 b and secondfluid outlet port 335 b. Second fluid channel 330 b may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Second fluid outlet port 335 b may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The second fluid channel and second fluid outlet port may beconfigured to dispense any solution described herein. The second fluidchannel and second fluid outlet port may be regarded as a secondprocessing bay or processing station. The second processing bay may beconfigured to perform a second processing operation, such as dispensingof a second solution to any of the first, second, third, fourth, orfifth substrates.

The system may further comprise third fluid channel 330 c and thirdfluid outlet port 335 c. Third fluid channel 330 c may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Third fluid outlet port 335 c may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The third fluid channel and third fluid outlet port may beconfigured to dispense any solution described herein. The third fluidchannel and third fluid outlet port may be regarded as a thirdprocessing bay or processing station. The third processing bay may beconfigured to perform a third processing operation, such as dispensingof a third solution to any of the first, second, third, fourth, or fifthsubstrates.

The system may further comprise fourth fluid channel 330 d and fourthfluid outlet port 335 d. Fourth fluid channel 330 d may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Fourth fluid outlet port 335 d may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The fourth fluid channel and fourth fluid outlet port may beconfigured to dispense any solution described herein. The fourth fluidchannel and fourth fluid outlet port may be regarded as a fourthprocessing bay or processing station. The fourth processing bay may beconfigured to perform a fourth processing operation, such as dispensingof a fourth solution to any of the first, second, third, fourth, orfifth substrates.

The system may further comprise a scanning optical imaging objective1110. The optical imaging objective may be regarded as a fifthprocessing bay or processing station.

The system may further comprise a moving arm 1220 g. The moving arm maybe configured to move laterally 1210 g or rotate 1215 g. The moving armmay be configured to move any of the first, second, third, fourth, orfifth substrates between different processing stations (such as bypicking up substrates and moving them to new locations). For instance,at a first point in time, the first substrate may undergo a firstoperation (such as dispensing of a first solution) at the firstprocessing bay, the second substrate may undergo a second operation(such as dispensing of a second solution) at the second processing bay,the third substrate may undergo a third operation (such as dispensing ofa third solution) at the first processing bay, the fourth substrate mayundergo a fourth operation (such as dispensing of a fourth solution) atthe fourth processing bay, and the fifth substrate may be imaged at thefifth processing bay. Upon completion of one or more of the first,second, third, or fourth operations, or of imaging, the moving arm maymove one or more of the first, second, third, fourth, or fifthsubstrates to one or more of the first, second, third, fourth, or fifthprocessing bays, where another operation may be completed. The patternof completing one or more operations and moving one or more substratesto another processing bay to complete another operation may be repeated,allowing rapid sequencing of the nucleic acids attached to the first,second, third, fourth, and fifth substrates using the systems andmethods described herein. The alternating pattern of dispensing andimaging may speed up the sequencing by increasing the duty cycle of theimaging process or the solution dispensing process.

Though depicted as comprising five substrates, four fluid channels, fourfluid outlet ports, and one optical imaging objective in FIG. 12G,system 1200 g may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The moving arm maymove so as to place any substrate under any fluid channel, fluid outletport, or optical imaging objective at any time.

FIG. 12H shows an architecture for a system 1200 h comprising aplurality of imaging heads scanning with shared translation androtational axes and independently rotating fields. The system maycomprise first and second read heads 1005 and 1015, respectively,configured to image substrate 310. The first and second read heads maybe similar to any read head described herein (such as with respect toFIG. 10). At a particular point in time, the first and second read headsmay be configured to image first and second paths 1010 and 1020,respectively. The first and second paths may be similar to any pathsdescribed herein (such as with respect to FIG. 10). The first and secondread heads may be configured to move 1210 h in a substantially radialdirection over the spinning substrate, thereby scanning the substrate.In the event that either the first or second read head does not moveprecisely radially, an image field or sensor of the read head may rotateto maintain a substantially tangential scan direction. A field rotationmay be accomplished using rotating prisms.

Though depicted as comprising two read heads and two imaging paths inFIG. 12H, system 1200 h may comprise any number of read heads or imagingpaths. For instance, the system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 read heads. The system may comprise at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 imaging paths.

FIG. 12I shows an architecture for a system 1200 i comprising multiplespindles scanning with a shared optical detection system. The system maycomprise first and second substrates 310 a and 310 b, respectively. Thefirst and second substrates may be similar to substrate 310 describedherein. The first and second substrates may be affixed to first andsecond spindles, respectively. The first and second spindles may impartrotational motion to the first and second substrates, respectively. Thesystem may comprise first and second optical imaging objectives 1110 aand 1110 b, respectively. The first and second optical imagingobjectives may be similar to optical imaging objective 1110 describedherein. The first and second optical imaging objectives may beconfigured to collect light from the first and second substrates,respectively. The first and second optical imaging objectives may passlight collected from the first and second substrates, respectively, tofirst and second mirrors 1280 a and 1280 b, respectively. In some cases,only one of the first and second optical imaging objective will collectlight at a particular instance in time.

The first and second mirrors may pass the light to a shared movablemirror. When in a first configuration 1285 a, the shared movable mirrormay direct light from the first substrate to a beamsplitter 1295. Thebeamsplitter may comprise a dichroic mirror. The beamsplitter may passlight to a detector 370, allowing the first substrate to be imaged. Thefirst substrate may be configured to be translated 1210 i, allowingdifferent locations on the first substrate to be imaged.

When in a second configuration 1285 b, the shared movable mirror maydirect light from the second substrate to the beamsplitter 1295. Thebeamsplitter may pass light to a detector 370, allowing the secondsubstrate to be imaged. The second substrate may be configured to betranslated 1210 i, allowing different locations on the second substrateto be imaged. Thus, by moving the movable mirror, the first and secondsubstrates may be imaged by a shared optical system.

The system may further comprise an excitation light source 1290. Thelight source may be configured to provide excitation light (such as forfluorescence imaging) to the first or second substrate. The excitationlight may be selectively delivered to the first or second substrateusing the movable mirror in a similar manner as for detection describedherein.

Though depicted as comprising two substrates, two imaging opticalobjectives, and two mirrors in FIG. 12I, system 1200 i may comprise anynumber of substrates, imaging optical objectives, or mirrors. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. The system may comprise at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, or at least 10 imaging optical objectives. The system maycomprise at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, or at least 10 mirrors.

FIG. 12H shows an architecture for a system comprising a plurality ofimaging heads scanning with shared translation and rotational axes andindependently rotating fields;

FIG. 12I shows an architecture for a system comprising multiple spindlesscanning with a shared optical detection system

FIG. 13 shows an architecture for a system 1300 comprising a pluralityof rotating spindles. The system 1300 may comprise substrate 310described herein. The substrate may be configured to rotate, asdescribed herein. The system may further comprise fluid channel 330 andfluid outlet port 335 described herein, or any other fluid channel andfluid outlet port described herein. The fluid channel and fluid outletport may be configured to dispense any solution described herein. Thefluid channel and fluid outlet port may be configured to move 1315 arelative to the substrate. For instance, the fluid channel and fluidoutlet port may be configured to move to a position above (such as nearthe center of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The fluidchannel and fluid outlet port may be configured to move to a positionaway from the substrate during the period in which the fluid channel andfluid outlet port are not dispensing a solution. The system may furthercomprise optical imaging objective 1110 described herein. The opticalimaging objective may be configured to move 1310 a relative for thesubstrate. For instance, the optical imaging objective may be configuredto move to a position above (such as near the center of, or radiallyscanning) the substrate during periods of time in which the substrate isbeing imaged. The optical imaging objective may be configured to move toa position away from the substrate during the period in which thesubstrate is not being imaged.

The system may further comprise a first spindle 1305 a and a secondspindle 1305 b. The first spindle may be interior to the second spindle.The first spindle may be exterior to the second spindle. The secondspindle may be interior to the first spindle. The second spindle may beexterior to the first spindle. The first and second spindles may each beconfigured to rotate independently of each other. The first and secondspindles may be configured to rotate with different angular velocities.For instance, the first spindle may be configured to rotate with a firstangular velocity and the second spindle may be configured to rotate witha second angular velocity. The first angular velocity may be less thanthe second angular velocity. The first spindle may be configured torotate at a relatively low angular velocity (such as an angular velocitybetween about 0 rpm and about 100 rpm) during periods in which asolution is being dispensed to the substrate. The second spindle may beconfigured to rotate at a relatively high angular velocity (such as anangular velocity between about 100 rpm and about 1,000 rpm) duringperiods in which the substrate is being imaged. Alternatively, thereverse may apply. The substrate may be transferred between the firstand second spindles to complete each of the dispensing and imagingoperations.

The system may comprise any number of spindles. For example, the systemmay comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or morespindles. Alternatively or in addition, the system may comprise at mostabout 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 spindle. A given spindle maybe interior or exterior relative to one or more other spindles in thesystem. In some instances, each of the spindles may rotate independentlyof each other. In some instances, at least a subset of the spindles mayrotate independently of each other. In some instances, at least a subsetof the spindles may rotate dependently of each other (e.g.,simultaneously at the same angular velocity). The spindles may rotatewith respect to the same axis or different axes. In some instances, eachspindle may rotate with different angular velocities. In some instances,at least a subset of the spindles may rotate with different angularvelocities.

Though depicted as utilizing a moving fluid channel and optical imagingobjective in FIG. 13, the system 1300 may be configured in other mannersas described herein. For instance, the system may be configured suchthat the fluid channel and optical imaging objective are stationary andthe substrate is configured to move. The system may be configured in anyother manner described herein.

Application to Other Analytes

Though described herein as useful for sequencing nucleic acids, thesystems and method described herein may be applied to other analytesand/or other applications processing such analytes. FIG. 14 shows aflowchart for a method 1400 for processing an analyte.

In a first operation 1410, the method may comprise providing a substratecomprising a planar array having immobilized thereto an analyte, whereinthe substrate is configured to rotate with respect to an axis. The axismay be an axis through the center of the substrate. The axis may be anoff-center axis. The substrate may be any substrate described herein. Insome instances, the planar array may comprise a single type of analyte.In other instances, the planar array may comprise two or more types ofanalytes. The two or more types of analytes may be arranged randomly.The two or more types of analytes may be arranged in a regular pattern.The analyte may be any biological sample described herein or derivativethereof. For example, the analyte may be a single cell analyte. Theanalyte may be a nucleic acid molecule. The analyte may be a proteinmolecule. The analyte may be a single cell. The analyte may be aparticle. The analyte may be an organism. The analyte may be part of acolony. In some cases, the analyte may be or be derived from anon-biological sample. The analyte may be immobilized in an individuallyaddressable location on the planar array. The analyte may be immobilizedto the substrate via a linker configured to bind to the analyte. Forexample, the linker may comprise a carbohydrate molecule. The link maycomprise an affinity binding protein. The linker may be hydrophilic. Thelinker may be hydrophobic. The linker may be electrostatic. The linkermay be labeled. The linker may be integral to the substrate. The linkermay be an independent layer on the substrate.

In a second operation 1420, the method may comprise directing a solutioncomprising a plurality of adaptors across the planar array duringrotation of the substrate. The solution may comprise any solution orreagent described herein. The plurality of adaptors may be configured tointeract with the analyte immobilized to the planar array. For example,where the analyte is a nucleic acid molecule, the plurality of adaptorsmay comprise a plurality of probes. A given probe of the plurality ofprobes may comprise a random sequence or a targeted sequence, such as ahomopolymer sequence or a dibase or tribase repeating sequence. In someinstances, the probe may be a dibase probe. In some instances, the probemay be about 1 to 10 bases in length. In some instances, the probe maybe about 10 to 20 bases in length. In some instances, the probe may beat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, or more bases. Alternatively or incombination the probe may be at most about 50, 40, 30, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 base. In anotherexample, where the analyte is a protein molecule, the plurality ofadaptors may comprise a plurality of antibodies. A given antibody of theplurality of antibodies may have binding specificity to one or moretypes of proteins. In other instances, the plurality of adaptors maycomprise any combination of a plurality of oligonucleotide molecules,carbohydrate molecules, lipid molecules, affinity binding proteins,aptamers, antibodies, enzymes, or other reagents. The plurality ofadaptors may be hydrophilic. The plurality of adaptors may behydrophobic. The plurality of adaptors may be electrostatic. Theplurality of adaptors may be labeled. The plurality of adaptors maycomprise a mixture of labeled and unlabeled components. In someinstances, the plurality of adaptors may not be labeled.

In an operation 1430, the method may comprise subjecting the analyte toconditions sufficient to cause a reaction between the analyte and theplurality of adaptors. In an operation 1440, the method may comprisedetecting a signal indicative of the reaction between the analyte andthe plurality of adaptors, thereby analyzing the analyte.

The method may further comprise, prior to operation 1410, directing theanalyte across the substrate comprising the linker. For example, priorto or during dispensing of the analyte, the substrate may be rotated tocoat the substrate surface and/or the planar array with the analyte. Insome instances, the analyte may be coupled to a bead, which bead isimmobilized to the planar array.

The method may further comprise recycling, as described elsewhereherein, a subset of the solution that has contacted the substrate. Therecycling may comprise collecting, filtering, and reusing the subset ofthe solution. The filtering may comprise molecular filtering. Themolecular filtering may comprise specific nucleic acid filtering (i.e.filtering for a specific nucleic acid). The nucleic acid filtering maycomprise exposure of the solution to an array of oligonucleotideextension compounds which may specifically bind to contaminantnucleotides or nucleic acids.

The signal may be an optical signal. The signal may be a fluorescencesignal. The signal may be a light absorption signal. The signal may be alight scattering signal. The signal may be a luminescent signal. Thesignal may be a phosphorescence signal. The signal may be an electricalsignal. The signal may be an acoustic signal. The signal may be amagnetic signal. The signal may be any detectable signal. Alternativelyor in addition to the optical sensors described herein, the system maycomprise one or more other detectors (e.g., acoustic detector, etc.)configured to detect the detectable signal.

In some instances, the method may further comprise, prior to operation1420, subjecting the substrate to rotation with respect to the centralaxis.

In some instances, the method may further comprise terminating rotationof the substrate prior to detecting the signal in operation 1440. Inother instances, the signal may be detected in operation 1440 while thesubstrate is rotating.

The signal may be generated by binding of a label to the analyte. Thelabel may be bound to a molecule, particle, cell, or organism. The labelmay be bound to the molecule, particle, cell, or organism prior tooperation 1410. The label may be bound to the molecule, particle, cell,or organism subsequent to operation 1410. The signal may be generated byformation of a detectable product by a chemical reaction. The reactionmay comprise an enzymatic reaction. The signal may be generated byformation of a detectable product by physical association. The signalmay be generated by formation of a detectable product by proximityassociation. The proximity association may comprise Förster resonanceenergy transfer (FRET). The proximity association may compriseassociation with a complementation enzyme. The signal may be generatedby a single reaction. The signal may be generated by a plurality ofreactions. The plurality of reactions may occur in series. The pluralityof reactions may occur in parallel. The plurality of reactions maycomprise one or more repetitions of a reaction. For example, thereaction may comprise a hybridization reaction or ligation reaction. Thereaction may comprise a hybridization reaction and a ligation reaction.

The method may further comprise repeating operations 1420, 1430, and1440 one or more times. Different solutions may be directed to theplanar array during rotation of the substrate for consecutive cycles.

Many variations, alterations, and adaptations based on the method 1400provided herein are possible. For example, the order of the operationsof the method 1400 may be changed, some of the operations removed, someof the operations duplicated, and additional operations added asappropriate. Some of the operations may be performed in succession. Someof the operations may be performed in parallel. Some of the operationsmay be performed once. Some of the operations may be performed more thanonce. Some of the operations may comprise sub-operations. Some of theoperations may be automated. Some of the operations may be manual.

FIG. 15 shows a first example of a system 1500 for isolating an analyte.The system may comprise a plurality of linkers 1510 a, 1510 b, 1510 c,and 1510 d. The plurality of linkers may be adhered or otherwise affixedto substrate 310 described herein. For instance, each linker may bebound to a particular individually addressable location of the pluralityof individually addressable locations described herein. Linkers 1510 a,1510 b, 1510 c, and 1510 d may comprise any linker described herein.Some or all of linkers 1510 a, 1510 b, 1510 c, and 1510 d may be thesame. Some or all of linkers 1510 a, 1510 b, 1510 c, and 1510 d may bedifferent. The linkers may be configured to interact with analytes 1520a and 1520 b. For instance, the linkers may be configured to bind toanalytes 1520 a and 1520 b through any interaction described herein.Analytes 1520 a and 1520 b may comprise any analyte described herein.Analytes 1520 a and 1520 b may be the same. Analytes 1520 a n and 1520 bmay be different. The linkers may be configured to interact specificallywith particular analytes and/or types thereof. For instance, linker 1510b may be configured to interact specifically with analyte 1520 a. Linker1510 d may be configured to interact specifically with analyte 1520 b.Any linker may be configured to interact with any analyte. In thismanner, specific analytes may be bound to specific locations on thesubstrate. Though shown as comprising four linkers and two analytes inFIG. 15, system 1500 may comprise any number of linkers and analytes.For instance, system 1500 may comprise at least 1, at least 2, at least5, at least 10, at least 20, at least 50, at least 100, at least 200, atleast 500, at least 1,000, at least 2,000, at least 5,000, at least10,000, at least 20,000, at least 50,000, at least 100,000, at least200,000, at least 500,000, at least 1,000,000, at least 2,000,000, atleast 5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, at least 1,000,000,000 linkers, or a number of linkers thatis within a range defined by any two of the preceding values. System1500 may comprise at least 1, at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100, at least 200, at least 500, atleast 1,000, at least 2,000, at least 5,000, at least 10,000, at least20,000, at least 50,000, at least 100,000, at least 200,000, at least500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, atleast 10,000,000, at least 20,000,000, at least 50,000,000, at least100,000,000, at least 200,000,000, at least 500,000,000, at least1,000,000,000 analytes, or a number of analytes that is within a rangedefined by any two of the preceding values.

FIG. 16 shows a second example of a system 1600 for isolating ananalyte. The system may comprise a well configured to physically trap aparticle. The well may comprise an individually addressable location ofthe plurality of individually addressable locations described herein.The well may be configured to trap an analyte. For instance, the wellmay be configured to trap a droplet of blood 1630. For example, thedroplet of blood may comprise white blood cells 1640, red blood cells1650, and circulating tumor cells 1660. The well may be configured totrap any other analyte described herein. The well may be constructed inlayers using microfabrication materials and techniques. For instance,the well may comprise a base layer 1605. The base layer may comprisesilicon. The well may comprise an oxide layer 1610. The oxide layer maycomprise silicon oxide. The well may comprise a metal layer 1615. Themetal may comprise nickel or aluminum. The well may comprise a nanotubelayer 1620. The nanotube layer may comprise one or more carbonnanotubes. The well may comprise a confinement layer 1625. Theconfinement layer may comprise a photoresist. The photoresist maycomprise SU-8. The nanotube layer and confinement layer may beconfigured to together trap the cell.

FIG. 17 shows examples of control systems to compensate for velocitygradients during scanning. Such control system may algorithmicallycompensate for velocity gradients. The control system may predictive oradaptively compensate for tangential velocity gradients. In a firstcontrol system, illustrated on the left of FIG. 17, the control systemmay, based on scanning of a rotating substrate, measure residual(uncorrected) velocity errors during scanning, compute a compensationcorrection factor, and use the compensation correction factor to set (oradjust) a compensation factor to reduce the velocity errors forsubsequent scanning results. The first control system may be a closedloop control system that removes (or otherwise reduces) velocity errors.In a second control system, illustrated on the right of FIG. 17, thecontrol system may, based on knowledge of the geometry and relativeposition of the scanning relative to the substrate, directly compute (orpredict) the expected velocity gradient, and set (or adjust) the systemto remove the expected gradient.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 1 shows acomputer system 101 that is programmed or otherwise configured tosequence a nucleic acid sample. The computer system 101 can regulatevarious aspects of methods and systems of the present disclosure.

The computer system 101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 101 also includes memory or memorylocation 110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 115 (e.g., hard disk), communicationinterface 120 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 125, such as cache, other memory,data storage and/or electronic display adapters. The memory 110, storageunit 115, interface 120 and peripheral devices 125 are in communicationwith the CPU 105 through a communication bus (solid lines), such as amotherboard. The storage unit 115 can be a data storage unit (or datarepository) for storing data. The computer system 101 can be operativelycoupled to a computer network (“network”) 130 with the aid of thecommunication interface 120. The network 130 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 130 in some cases is atelecommunication and/or data network. The network 130 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 130, in some cases with the aid of thecomputer system 101, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 101 to behave as a clientor a server.

The CPU 105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 110. The instructionscan be directed to the CPU 105, which can subsequently program orotherwise configure the CPU 105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 105 can includefetch, decode, execute, and writeback.

The CPU 105 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 115 can store files, such as drivers, libraries andsaved programs. The storage unit 115 can store user data, e.g., userpreferences and user programs. The computer system 101 in some cases caninclude one or more additional data storage units that are external tothe computer system 101, such as located on a remote server that is incommunication with the computer system 101 through an intranet or theInternet.

The computer system 101 can communicate with one or more remote computersystems through the network 130. For instance, the computer system 101can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 101 via the network 130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 101, such as, for example, on the memory110 or electronic storage unit 115. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 105. In some cases, the code canbe retrieved from the storage unit 115 and stored on the memory 110 forready access by the processor 105. In some situations, the electronicstorage unit 115 can be precluded, and machine-executable instructionsare stored on memory 110.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 101, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 101 can include or be in communication with anelectronic display 135 that comprises a user interface (UI) 140 forproviding, for example, nucleic acid sequencing information to a user.Examples of UI's include, without limitation, a graphical user interface(GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 105.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for processing an biological analyte,comprising: (a) directing a solution comprising a plurality of probesfrom a dispensing unit to a fluid medium and from said fluid medium to afirst location and a second location of a substrate, wherein said firstlocation and said second location are disposed at different radialdistances from a central axis of said substrate; (b) coupling at leastone probe of said plurality of probes to said biological analyte; and(c) detecting one or more signals or signal changes from said biologicalanalyte having said at least one probe coupled thereto.
 2. The method ofclaim 1, wherein said fluid medium comprises air.
 3. The method of claim1, wherein said dispensing unit comprises a dispensing nozzle configuredto dispense said solution to both said first location and said secondlocation of said substrate.
 4. The method of claim 3, wherein saiddirecting in (a) comprises subjecting said substrate to movementrelative to said dispensing nozzle.
 5. The method of claim 4, whereinsaid movement comprises rotational motion of said substrate.
 6. Themethod of claim 4, wherein said movement comprises non-rotational motionof said substrate.
 7. The method of claim 1, wherein said dispensingunit comprises a first dispensing nozzle configured to dispense saidsolution to said first location and a second dispensing nozzleconfigured to dispense said solution to said second location.
 8. Themethod of claim 1, wherein said dispensing unit is configured to deliversaid solution to said substrate as a jet.
 9. The method of claim 1,wherein said dispensing unit is configured to deliver said solution tosaid substrate as a spray and/or in droplets.
 10. The method of claim 9,wherein said dispensing unit is configured to nebulize said solutionprior to delivery to said substrate.
 11. The method of claim 1, whereinsaid substrate is substantially planar.
 12. The method of claim 1,wherein said substrate is textured or patterned.
 13. The method of claim1, wherein said substrate comprises an array of individually addressablelocations, wherein said biological analyte is immobilized to anindividually addressable location of said array, and wherein said arrayhas immobilized thereto one or more additional biological analytes atdifferent individually addressable locations.
 14. The method of claim 1,wherein said biological analyte is coupled to a bead, which bead isimmobilized to said substrate.
 15. The method of claim 14, wherein saidbead comprises a plurality of biological analytes, including saidbiological analyte, attached thereto, and wherein said plurality ofbiological analytes have sequence homology to one another.
 16. Themethod of claim 1, wherein said substrate comprises at least 100,000binders, wherein a binder of said at least 100,000 binders immobilizessaid biological analyte adjacent to said substrate.
 17. The method ofclaim 1, wherein said biological analyte is a nucleic acid molecule. 18.The method of claim 17, further comprising, based at least in part onsaid one or more signals or signal changes, determining a sequence of atleast a portion of said nucleic acid molecule.
 19. The method of claim17, further comprising, based at least in part on said one or moresignals or signal changes, identifying a presence of a homopolymersequence in said nucleic acid molecule.
 20. The method of claim 17,wherein said plurality of probes comprises a plurality of nucleotides ornucleotide analogs, and wherein said at least one probe is at least onenucleotide or nucleotide analog of said plurality of nucleotides ornucleotide analogs.
 21. The method of claim 20, wherein (b) comprisesincorporating said at least one nucleotide or nucleotide analog into agrowing strand that is complementary to said nucleic acid molecule. 22.The method of claim 17, wherein said plurality of probes comprises aplurality of oligonucleotide molecules, and wherein said at least oneprobe is at least one oligonucleotide molecule of said plurality ofoligonucleotide molecules.
 23. The method of claim 22, wherein (b)comprises hybridizing said at least one oligonucleotide molecule to saidnucleic acid molecule.
 24. The method of claim 1, wherein said detectingin (c) comprises continuously scanning said substrate during relativemovement of said substrate with respect to a detection system configuredto perform said detecting.
 25. The method of claim 1, wherein said oneor more signals or signal changes comprise one or more optical signalsor optical signal changes.
 26. The method of claim 1, furthercomprising, subsequent to (c), (i) dispensing an additional solutioncomprising an additional plurality of probes to said substrate, whereinsaid additional plurality of probes is different from said plurality ofprobes, wherein at least one additional probe of said additionalplurality of probes couples to said biological analyte, and (ii)detecting one or more additional signals or additional signal changesfrom said biological analyte having said at least one additional probecoupled thereto, to analyze said biological analyte.
 27. The method ofclaim 26, wherein said plurality of probes comprises a first pluralityof nucleotides or nucleotide analogs of a first canonical base type andwherein said additional plurality of probes comprises a second pluralityof nucleotides or nucleotide analogs of a second canonical base typedifferent than said first canonical base type.
 28. The method of claim1, wherein said plurality of probes comprises a first plurality ofnucleotides or nucleotide analogs of a first canonical base type and asecond plurality of nucleotides or nucleotide analogs of a secondcanonical base type different than said first canonical base type. 29.The method of claim 1, wherein prior to (c), a substantially even layerof said solution is adjacent to said substrate.